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  • 1. Andersen, Birgit
    et al.
    Lundgren, Stina
    Dobritzsch, Doreen
    Karolinska Institutet.
    Piskur, Jure
    A recruited protease is involved in catabolism of pyrimidines2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 2, p. 243-250Article in journal (Refereed)
    Abstract [en]

    In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

  • 2. Andersen, G
    et al.
    Merico, A
    Björnberg, O
    Andersen, B
    Schnackerz, K D
    Dobritzsch, Doreen
    Karolinska Institutet.
    Piskur, J
    Compagno, C
    Catabolism of pyrimidines in yeast: a tool to understand degradation of anticancer drugs2006In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 25, no 9-11, p. 991-996Article in journal (Refereed)
    Abstract [en]

    The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides/antibiotics. During the last decade we have developed a yeast species, Saccharomyces kluyveri, as a model and tool to study the genes and enzymes of the pyrimidine catabolic pathway. In this report, we studied degradation of uracil and its putative degradation products in 38 yeasts and showed that this pathway was present in the ancient yeasts but was lost approximately 100 million years ago in the S. cerevisiae lineage.

  • 3. Andersen, Gorm
    et al.
    Andersen, Birgit
    Dobritzsch, Doreen
    Karolinska Institutet.
    Schnackerz, Klaus D
    Piskur, Jure
    A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast2007In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 7, p. 1804-1017Article in journal (Refereed)
    Abstract [en]

    In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.

  • 4.
    Bauer, Paul
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Amrein, Beat A.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, S. C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 12016In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

  • 5. Beck, Halfdan
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Piskur, Jure
    Saccharomyces kluyveri as a model organism to study pyrimidine degradation2008In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 8, no 8, p. 1209-1213Article, review/survey (Refereed)
    Abstract [en]

    The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.

  • 6. Claesson, Magnus
    et al.
    Siitonen, Vilja
    Dobritzsch, Doreen
    Karolinska Institutet.
    Metsä-Ketelä, Mikko
    Schneider, Gunter
    Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 17, p. 3251-3263Article in journal (Refereed)
    Abstract [en]

    The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

  • 7.
    Doak, Bradley C
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Zheng, Jie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    How Beyond Rule of 5 Drugs and Clinical Candidates Bind to Their Targets.2016In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 6, p. 2312-2327Article in journal (Refereed)
    Abstract [en]

    To improve discovery of drugs for difficult targets, the opportunities of chemical space beyond the rule of 5 (bRo5) were examined by retrospective analysis of a comprehensive set of structures for complexes between drugs and clinical candidates and their targets. The analysis illustrates the potential of compounds far beyond rule of 5 space to modulate novel and difficult target classes that have large, flat, and groove-shaped binding sites. However, ligand efficiencies are significantly reduced for flat- and groove-shape binding sites, suggesting that adjustments of how to use such metrics are required. Ligands bRo5 appear to benefit from an appropriate balance between rigidity and flexibility to bind with sufficient affinity to their targets, with macrocycles and nonmacrocycles being found to have similar flexibility. However, macrocycles were more disk- and spherelike, which may contribute to their superior binding to flat sites, while rigidification of nonmacrocycles lead to rodlike ligands that bind well to groove-shaped binding sites. These insights should contribute to altering perceptions of what targets are considered "druggable" and provide support for drug design in beyond rule of 5 space.

  • 8.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Andersen, Birgit
    Piskur, Jure
    Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri2005In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 61, no Pt 4, p. 359-362Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.

  • 9.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Gojković, Zoran
    Andersen, Birgit
    Piskur, Jure
    Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, no Pt 7, p. 1267-1269Article in journal (Refereed)
    Abstract [en]

    In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.

  • 10.
    Dobritzsch, Doreen
    et al.
    Martin-Luther-Universität Halle-Wittenberg.
    König, S
    Schneider, G
    Lu, G
    High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis: Implications for substrate activation in pyruvate decarboxylases1998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 32, p. 20196-20204Article in journal (Refereed)
    Abstract [en]

    The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

  • 11.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Lindh, Ingrid
    Uysal, Hüseyin
    Nandakumar, Kutty S
    Burkhardt, Harald
    Schneider, Gunter
    Holmdahl, Rikard
    Crystal structure of an arthritogenic anticollagen immune complex2011In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 63, no 12, p. 3740-3748Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation.

    METHODS: Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide.

    RESULTS: The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope.

    CONCLUSION: The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures.

  • 12.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Persson, K
    Schneider, G
    Lindqvist, Y
    Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase2001In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 57, no Pt 1, p. 153-155Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.

  • 13.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Ricagno, Stefano
    Schneider, Gunter
    Schnackerz, Klaus D
    Lindqvist, Ylva
    Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil: Implications for mechanism of inhibition and electron transfer2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 15, p. 13155-13166Article in journal (Refereed)
    Abstract [en]

    Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.

  • 14.
    Dobritzsch, Doreen
    et al.
    Karolinska Institutet.
    Schneider, G
    Schnackerz, K D
    Lindqvist, Y
    Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4, p. 650-660Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5-fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2x 111 kDa) determined at 1.9 A resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe-4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe-S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5-fluorouracil reveals the architecture of the substrate-binding sites and residues responsible for recognition and binding of the drug.

  • 15.
    Dobritzsch, Doreen
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Wang, Huaming
    Schneider, Gunter
    Yu, Shukun
    Structural and functional characterization of ochratoxinase, a novel mycotoxin-degrading enzyme2014In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 462, no 3, p. 441-452Article in journal (Refereed)
    Abstract [en]

    Ochratoxin, with ochratoxin A as the dominant form, is one of the five major mycotoxins most harmful to humans and animals. It is produced by Aspergillus and Penicillium species and occurs in a wide range of agricultural products. Detoxification of contaminated food is a challenging health issue. In the present paper we report the identification, characterization and crystal structure (at 2.2 angstrom) of a novel microbial ochratoxinase from Aspergillus niger. A putative amidase gene encoding a 480 amino acid polypeptide was cloned and homologously expressed in A. niger. The recombinant protein is N-terminally truncated, thermostable, has optimal activity at pH similar to 6 and 66 degrees C, and is more efficient in ochratoxin A hydrolysis than carboxypeptidase A and Y, the two previously known enzymes capable of degrading this mycotoxin. The subunit of the homo-octameric enzyme folds into a two-domain structure characteristic of a metal dependent amidohydrolase, with a twisted TIM (triosephosphateisomerase)-barrel and a smaller beta-sandwich domain. The active site contains an aspartate residue for acid base catalysis, and a carboxylated lysine and four histidine residues for binding of a binuclear metal centre.

  • 16.
    Ge, Changrong P
    et al.
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Tong, Dongmei R
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Southern Med Univ, Dept Pathophysiol, Key Lab Shock & Microcirculat Res Guangdong, Guangzhou, Guangdong, Peoples R China..
    Liang, Bibo T
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Southern Med Univ, Dept Pathophysiol, Key Lab Shock & Microcirculat Res Guangdong, Guangzhou, Guangdong, Peoples R China..
    Lönnblom, Erik S
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden..
    Schneider, Nadine K
    Goethe Univ, Fraunhofer Inst Mol Biol & Appl Ecol IME, Project Grp Translat Med & Pharmacol, Frankfurt, Germany.;Goethe Univ, Div Rheumatol, Univ Hosp Frankfurt, Frankfurt, Germany..
    Hagert, Cecilia U
    Univ Turku, Medicity Res Lab, Turku, Finland.;Natl Doctoral Programme Informat & Struct Biol, Turku, Finland..
    Viljanen, Johan V.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Ayoglu, Burcu R
    KTH Royal Inst Technol, Sch Biotechnol, Sci Life Lab, Affin Prote, Stockholm, Sweden..
    Stawikowska, Roma T
    Florida Atlantic Univ, Dept Chem & Biochem, Jupiter, FL USA..
    Nilsson, Peter C
    KTH Royal Inst Technol, Sch Biotechnol, Sci Life Lab, Affin Prote, Stockholm, Sweden..
    Fields, Gregg B
    Florida Atlantic Univ, Dept Chem & Biochem, Jupiter, FL USA..
    Skogh, Thomas A
    Linkoping Univ, Dept Rheumatol, Dept Clin & Expt Med, Linkoping, Sweden..
    Kastbom, Alf R
    Linkoping Univ, Dept Rheumatol, Dept Clin & Expt Med, Linkoping, Sweden..
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.
    Burkhardt, Harald T
    Goethe Univ, Fraunhofer Inst Mol Biol & Appl Ecol IME, Project Grp Translat Med & Pharmacol, Frankfurt, Germany.;Goethe Univ, Div Rheumatol, Univ Hosp Frankfurt, Frankfurt, Germany..
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Holmdahl, Rikard K
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, Stockholm, Sweden.;Univ Turku, Medicity Res Lab, Turku, Finland.;Natl Doctoral Programme Informat & Struct Biol, Turku, Finland.;Southern Med Univ, Ctr Med Immunopharmacol Res, Guangzhou, Guangdong, Peoples R China..
    Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage2017In: JCI INSIGHT, ISSN 2379-3708, Vol. 2, no 13, article id e93688Article in journal (Refereed)
    Abstract [en]

    Today, it is known that autoimmune diseases start a long time before clinical symptoms appear. Anti-citrullinated protein antibodies (ACPAs) appear many years before the clinical onset of rheumatoid arthritis (RA). However, it is still unclear if and how ACPAs are arthritogenic. To better understand the molecular basis of pathogenicity of ACPAs, we investigated autoantibodies reactive against the C1 epitope of collagen type II (CII) and its citrullinated variants. We found that these antibodies are commonly occurring in RA. A mAb (ACC1) against citrullinated C1 was found to cross-react with several noncitrullinated epitopes on native CII, causing proteoglycan depletion of cartilage and severe arthritis in mice. Structural studies by X-ray crystallography showed that such recognition is governed by a shared structural motif "RG-TG" within all the epitopes, including electrostatic potential-controlled citrulline specificity. Overall, we have demonstrated a molecular mechanism that explains how ACPAs trigger arthritis.

  • 17.
    Ge, Changrong
    et al.
    Karolinska Inst, Stockholm, Sweden.
    Xu, Bingze
    Karolinska Inst, Stockholm, Sweden.
    Liang, Bibo
    Karolinska Inst, Stockholm, Sweden;Southern Med Univ, Guangzhou, Guangdong, Peoples R China.
    Lönnblom, Erik
    Karolinska Inst, Stockholm, Sweden.
    Lundström, Susanna L.
    Karolinska Inst, Stockholm, Sweden.
    Zubarev, Roman A.
    Karolinska Inst, Stockholm, Sweden.
    Ayoglu, Burcu
    KTH Royal Inst Technol, Stockholm, Sweden.
    Nilsson, Peter
    KTH Royal Inst Technol, Stockholm, Sweden.
    Skogh, Thomas
    Linkoping Univ, Linkoping, Sweden.
    Kastbom, Alf
    Linkoping Univ, Linkoping, Sweden.
    Malmström, Vivianne
    Karolinska Inst, Stockholm, Sweden;Karolinska Univ Hosp, Stockholm, Sweden.
    Klareskog, Lars
    Karolinska Inst, Stockholm, Sweden;Karolinska Univ Hosp, Stockholm, Sweden.
    Toes, Rene E. M.
    Leiden Univ, Med Ctr, Leiden, Netherlands.
    Rispens, Theo
    Univ Amsterdam, Amsterdam, Netherlands.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Holmdahl, Rikard
    Karolinska Inst, Stockholm, Sweden;Southern Med Univ, Guangzhou, Guangdong, Peoples R China.
    Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies2019In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 210-221Article in journal (Refereed)
    Abstract [en]

    Objective: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA.

    Methods: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients.

    Results: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients.

    Conclusion: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

  • 18. Haag, Sabrina
    et al.
    Tuncel, Jonatan
    Thordardottir, Soley
    Mason, Daniel E.
    Yau, Anthony C. Y.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Backlund, Johan
    Peters, Eric C.
    Holmdahl, Rikard
    Positional Identification of RT1-B (HLA-DQ) as Susceptibility Locus for Autoimmune Arthritis2015In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 194, no 6, p. 2539-2550Article in journal (Refereed)
    Abstract [en]

    Rheumatoid arthritis (RA) is associated with amino acid variants in multiple MHC molecules. The association to MHC class II (MHC-II) has been studied in several animal models of RA. In most cases these models depend on T cells restricted to a single immunodominant peptide of the immunizing Ag, which does not resemble the autoreactive T cells in RA. An exception is pristane-induced arthritis (PIA) in the rat where polyclonal T cells induce chronic arthritis after being primed against endogenous Ags. In this study, we used a mixed genetic and functional approach to show that RT1-Ba and RT1-Bb (RT1-B locus), the rat orthologs of HLA-DQA and HLA-DQB, determine the onset and severity of PIA. We isolated a 0.2-Mb interval within the MHC-II locus of three MHC-congenic strains, of which two were protected from severe PIA. Comparison of sequence and expression variation, as well as in vivo blocking of RT1-B and RT1-D (HLA-DR), showed that arthritis in these strains is regulated by coding polymorphisms in the RT1-B genes. Motif prediction based on MHC-II eluted peptides and structural homology modeling suggested that variants in the RT1-B P1 pocket, which likely affect the editing capacity by RT1-DM, are important for the development of PIA.

  • 19.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ntuku, Siphosethu
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Oliveira, Ana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer2017In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed)
    Abstract [en]

    Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes.

  • 20.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chu, Thao
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Löfgren, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin2018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, p. 1059-1062Article in journal (Refereed)
    Abstract [en]

    Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.

  • 21.
    Hampel, Sabrina
    et al.
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Steitz, Jan-Patrick
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Baierl, Anna
    Forschungszentrum Julich, IBG Biotechnol 1, Wilhelm Johnen Str, D-52425 Julich, Germany.
    Lehwald, Patrizia
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Wiesli, Luzia
    Empa Swiss Fed Labs Mat Sci & Technol, Lab Biointerfaces, Lerchenfeldstr 5, CH-9014 St Gallen, Switzerland.
    Richter, Michael
    Empa Swiss Fed Labs Mat Sci & Technol, Lab Biointerfaces, Lerchenfeldstr 5, CH-9014 St Gallen, Switzerland;Fraunhofer Inst Interfacial Engn & Biotechnol IGB, Branch BioCat, Schulgasse 11a, D-94315 Straubing, Germany.
    Fries, Alexander
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Pohl, Martina
    Forschungszentrum Julich, IBG Biotechnol 1, Wilhelm Johnen Str, D-52425 Julich, Germany.
    Schneider, Gunter
    Karolinska Inst, Dept Med Biochem & Biophys, Tomtebodavagen 6, S-17177 Stockholm, Sweden.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mueller, Michael
    Albert Ludwigs Univ Freiburg, Inst Pharmazeut Wissensch, Albertstr 25, D-79104 Freiburg, Germany.
    Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 21, p. 2283-2292Article in journal (Refereed)
    Abstract [en]

    A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55 angstrom.

  • 22.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme2018In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed)
    Abstract [en]

    The epoxide hydrolase StEH1 catalyzes the hydrolysis of trans-methylstyrene oxide to 1-phenyl­propane-1,2-diol. The (S,S)-epoxide is exclusively transformed into the (1R,2S)-diol, while hydrolysis of the (R,R)-epoxide results in a mixture of product enantiomers. In order to understand the differences in the stereoconfigurations of the products, the reactions were studied kinetically during both the pre-steady-state and steady-state phases. A number of closely related StEH1 variants were analyzed in parallel, and the results were rationalized by structure–activity analysis using the available crystal structures of all tested enzyme variants. Finally, empirical valence-bond simulations were performed in order to provide additional insight into the observed kinetic behaviour and ratios of the diol product enantiomers. These combined data allow us to present a model for the flux through the catalyzed reactions. With the (R,R)-epoxide, ring opening may occur at either C atom and with similar energy barriers for hydrolysis, resulting in a mixture of diol enantiomer products. However, with the (S,S)-epoxide, although either epoxide C atom may react to form the covalent enzyme intermediate, only the pro-(R,S) alkylenzyme is amenable to subsequent hydrolysis. Previously contradictory observations from kinetics experiments as well as product ratios can therefore now be explained for this biocatalytically relevant enzyme.

  • 23. Koskiniemi, Hanna
    et al.
    Metsä-Ketelä, Mikko
    Dobritzsch, Doreen
    Karolinska Institutet.
    Kallio, Pauli
    Korhonen, Hanna
    Mäntsälä, Pekka
    Schneider, Gunter
    Niemi, Jarmo
    Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 372, no 3, p. 633-648Article in journal (Refereed)
    Abstract [en]

    Angucyclines are aromatic polyketides produced in Streptomycetes via complex enzymatic biosynthetic pathways. PgaE and CabE from S. sp PGA64 and S. sp. H021 are two related homo-dimeric FAD and NADPH dependent aromatic hydroxylases involved in the early steps of the angucycline core modification. Here we report the three-dimensional structures of these two enzymes determined by X-ray crystallography using multiple anomalous diffraction and molecular replacement, respectively, to resolutions of 1.8 A and 2.7 A. The enzyme subunits are built up of three domains, a FAD binding domain, a domain involved in substrate binding and a C-terminal thioredoxin-like domain of unknown function. The structure analysis identifies PgaE and CabE as members of the para-hydroxybenzoate hydroxylase (pHBH) fold family of aromatic hydroxylases. In contrast to phenol hydroxylase and 3-hydroxybenzoate hydroxylase that utilize the C-terminal domain for dimer formation, this domain is not part of the subunit-subunit interface in PgaE and CabE. Instead, dimer assembly occurs through interactions of their FAD binding domains. FAD is bound non-covalently in the "in"-conformation. The active sites in the two enzymes differ significantly from those of other aromatic hydroxylases. The volumes of the active site are significantly larger, as expected in view of the voluminous tetracyclic angucycline substrates. The structures further suggest that substrate binding and catalysis may involve dynamic rearrangements of the middle domain relative to the other two domains. Site-directed mutagenesis studies of putative catalytic groups in the active site of PgaE argue against enzyme-catalyzed substrate deprotonation as a step in catalysis. This is in contrast to pHBH, where deprotonation/protonation of the substrate has been suggested as an essential part of the enzymatic mechanism.

  • 24. Kuilenburg, André B P van
    et al.
    Meijer, Judith
    Tanck, Michael W T
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Zoetekouw, Lida
    Dekkers, Lois-Lee
    Roelofsen, Jeroen
    Meinsma, Rutger
    Wymenga, Machteld
    Kulik, Wim
    Büchel, Barbara
    Hennekam, Raoul C M
    Largiadèr, Carlo R
    Phenotypic and clinical implications of variants in the dihydropyrimidine dehydrogenase gene.2016In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1862, no 4, p. 754-762Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil, thymine and the antineoplastic agent 5-fluorouracil. Genetic variations in the gene encoding DPD (DPYD) have emerged as predictive risk alleles for 5FU-associated toxicity. Here we report an in-depth analysis of genetic variants in DPYD and their consequences for DPD activity and pyrimidine metabolites in 100 Dutch healthy volunteers. 34 SNPs were detected in DPYD and 15 SNPs were associated with altered plasma concentrations of pyrimidine metabolites. DPD activity was significantly associated with the plasma concentrations of uracil, the presence of a specific DPYD mutation (c.1905+1G>A) and the combined presence of three risk variants in DPYD (c.1905+1G>A, c.1129-5923C>G, c.2846A>T), but not with an altered uracil/dihydrouracil (U/UH2) ratio. Various haplotypes were associated with different DPD activities (haplotype D3, a decreased DPD activity; haplotype F2, an increased DPD activity). Functional analysis of eight recombinant mutant DPD enzymes showed a reduced DPD activity, ranging from 35% to 84% of the wild-type enzyme. Analysis of a DPD homology model indicated that the structural effect of the novel p.G401R mutation is most likely minor. The clinical relevance of the p.D949V mutation was demonstrated in a cancer patient heterozygous for the c.2846A>T mutation and a novel nonsense mutation c.1681C>T (p.R561X), experiencing severe grade IV toxicity. Our studies showed that the endogenous levels of uracil and the U/UH2 ratio are poor predictors of an impaired DPD activity. Loading studies with uracil to identify patients with a DPD deficiency warrants further investigation.

  • 25. Lindgren, Cecilia
    et al.
    Andersson, Ida E.
    Berg, Lotta
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ge, Changrong
    Haag, Sabrina
    Uciechowska, Urszula
    Holmdahl, Rikard
    Kihlberg, Jan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
    Linusson, Anna
    Hydroxyethylene isosteres introduced in type II collagen fragments substantially alter the structure and dynamics of class II MHC A(q)/glycopeptide complexes2015In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, no 22, p. 6203-6216Article in journal (Refereed)
    Abstract [en]

    Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the beta(1)-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response.

  • 26. Lohkamp, Bernhard
    et al.
    Andersen, Birgit
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum2006In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 62, no Pt 1, p. 36-38Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 A resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 A and one molecule in the asymmetric unit.

  • 27. Lohkamp, Bernhard
    et al.
    Andersen, Birgit
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 19, p. 13762-13776Article in journal (Refereed)
    Abstract [en]

    In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.

  • 28. Lohkamp, Bernhard
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    A mixture of fortunes: the curious determination of the structure of Escherichia coli BL21 Gab protein2008In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 64, no Pt 4, p. 407-415Article in journal (Refereed)
    Abstract [en]

    In protein crystallography, monodisperse protein samples of high purity are usually required in order to obtain diffraction-quality crystals. Here, crystals were reproducibly grown from a protein sample before its homogeneity had been determined. The sample was obtained after the first attempt to purify a recombinant target protein from an Escherichia coli cell lysate. Subsequent analysis revealed that it was a mixture of about 50 different proteins with no predominant species. Diffraction data were collected to 2.1 A and the space group was identified as I422. A molecular-replacement search with models of the expected target did not give a solution, which suggested that a contaminating E. coli protein had been crystallized. A PDB search revealed 256 structures determined in space group I422, of which 14 are E. coli proteins and two have unit-cell parameters similar to those observed. Molecular replacement with these structures showed a clear solution for one of them, the Gab protein. The structure is presented and compared with the deposited structure, from which it shows small but significant differences. The refined model contains bicine and sulfate as bound ligands, which provide insights into possible substrate-binding sites.

  • 29. Lohkamp, Bernhard
    et al.
    Voevodskaya, Nina
    Lindqvist, Ylva
    Dobritzsch, Doreen
    Karolinska Institutet.
    Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination2010In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1804, no 12, p. 2198-2206Article in journal (Refereed)
    Abstract [en]

    In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.

  • 30. Lu, G
    et al.
    Dobritzsch, Doreen
    Martin-Luther-Universität Halle-Wittenberg.
    Baumann, S
    Schneider, G
    König, S
    The structural basis of substrate activation in yeast pyruvate decarboxylase: A crystallographic and kinetic study2000In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 267, no 3, p. 861-868Article in journal (Refereed)
    Abstract [en]

    The crystal structure of the complex of the thiamine diphosphate dependent tetrameric enzyme pyruvate decarboxylase (PDC) from brewer's yeast strain with the activator pyruvamide has been determined to 2.4 A resolution. The asymmetric unit of the crystal contains two subunits, and the tetrameric molecule is generated by crystallographic symmetry. Structure analysis revealed conformational nonequivalence of the active sites. One of the two active sites in the asymmetric unit was found in an open conformation, with two active site loop regions (residues 104-113 and 290-304) disordered. In the other subunit, these loop regions are well-ordered and shield the active site from the bulk solution. In the closed enzyme subunit, one molecule of pyruvamide is bound in the active site channel, and is located in the vicinity of the thiazolium ring of the cofactor. A second pyruvamide binding site was found at the interface between the Pyr and the R domains of the subunit in the closed conformation, about 10 A away from residue C221. This second pyruvamide molecule might function in stabilizing the unique orientation of the R domain in this subunit which in turn is important for dimer-dimer interactions in the activated tetramer. No difference electron density in the close vicinity of the side chain of residue C221 was found, indicating that this residue does not form a covalent adduct with an activator molecule. Kinetic experiments showed that substrate activation was not affected by oxidation of cysteine residues and therefore does not seem to be dependent on intact thiol groups in the enzyme. The results suggest that a disorder-order transition of two active-site loop regions is a key event in the activation process triggered by the activator pyruvamide and that covalent modification of C221 is not required for this transition to occur. Based on these findings, a possible mechanism for the activation of PDC by its substrate, pyruvate, is proposed.

  • 31.
    Lu, G
    et al.
    Karolinska Institutet.
    Dobritzsch, Doreen
    Martin-Luther Universität Halle-Wittenberg.
    König, S
    Martin-Luther-Universität Halle-Wittenberg.
    Schneider, G
    Karolinska Institutet.
    Novel tetramer assembly of pyruvate decarboxylase from brewer's yeast observed in a new crystal form1997In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 403, no 3, p. 249-253Article in journal (Refereed)
    Abstract [en]

    A new crystal form of thiamine diphosphate dependent pyruvate decarboxylase from Saccharomyces cerevisiae has been obtained in the presence of the activator pyruvamide. The crystallographic structure analysis reveals differences in the domain packing in the enzyme subunit and a novel assembly of the subunits in the tetramer, when compared to the structure of native PDC. The orientation of the beta domains in the subunit differs by a 6.3 degrees and 8.3 degrees rotation, respectively, whereas the subunit-subunit interface in the dimer, formed by the alpha and gamma domains, is essentially maintained. In the tetramer, one of the dimers rotates relative to the second dimer by approximately 30 degrees creating a new dimer-dimer interface.

  • 32. Lundgren, Stina
    et al.
    Andersen, Birgit
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 49, p. 36037-36047Article in journal (Refereed)
    Abstract [en]

    Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.

  • 33. Lundgren, Stina
    et al.
    Andersen, Birgit
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    Crystallization and preliminary X-ray data analysis of beta-alanine synthase from Drosophila melanogaster2007In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 63, no Pt 10, p. 874-877Article in journal (Refereed)
    Abstract [en]

    Beta-alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 A at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 A, beta = 125.8 degrees) and contain 8-10 molecules per asymmetric unit.

  • 34. Lundgren, Stina
    et al.
    Gojković, Zoran
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases2003In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 51, p. 51851-51862Article in journal (Refereed)
    Abstract [en]

    beta-Alanine synthase (beta AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, beta ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of beta AS from Saccharomyces kluyveri to a resolution of 2.7 A. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of beta AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed alpha/beta-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by beta AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.

  • 35. Lundgren, Stina
    et al.
    Lohkamp, Bernhard
    Andersen, Birgit
    Piskur, Jure
    Dobritzsch, Doreen
    Karolinska Institutet.
    The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 377, no 5, p. 1544-1559Article in journal (Refereed)
    Abstract [en]

    Beta-alanine synthase (betaAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate to the corresponding beta-amino acids. betaASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote betaAS subfamily, we determined the crystal structure of Drosophila melanogaster betaAS to 2.8 A resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central beta-sandwich with a layer of alpha-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster betaAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.

  • 36.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bernhard, Lohkamp
    Krumpel, Michael
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Characterization and structure determination of human β-ureidopropionase reveal pH-dependent regulation by ligand-induced changes in oligomerizationManuscript (preprint) (Other academic)
  • 37.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lohkamp, Bernhard
    Krumpel, Michael
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism2018In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 475, no 14, p. 2395-2416Article in journal (Refereed)
    Abstract [en]

    β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human βUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer–dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly βUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer–dimer interface, effectively positioning the catalytically important Glu207 in the active site.

  • 38. Nakajima, Yoko
    et al.
    Meijer, Judith
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ito, Tetsuya
    Meinsma, Rutger
    Abeling, Nico G. G. M.
    Roelofsen, Jeroen
    Zoetekouw, Lida
    Watanabe, Yoriko
    Tashiro, Kyoko
    Lee, Tomoko
    Takeshima, Yasuhiro
    Mitsubuchi, Hiroshi
    Yoneyama, Akira
    Ohta, Kazuhide
    Eto, Kaoru
    Saito, Kayoko
    Kuhara, Tomiko
    van Kuilenburg, Andre B. P.
    Clinical, biochemical and molecular analysis of 13 Japanese patients with beta-ureidopropionase deficiency demonstrates high prevalence of the c.977G > A (p.R326Q) mutation2014In: Journal of Inherited Metabolic Disease, ISSN 0141-8955, E-ISSN 1573-2665, Vol. 37, no 5, p. 801-812Article in journal (Refereed)
    Abstract [en]

    beta-ureidopropionase (beta UP) deficiency is an autosomal recessive disease characterized by N-carbamyl-beta-amino aciduria. To date, only 16 genetically confirmed patients with beta UP deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 13 Japanese beta UP deficient patients. In this group of patients, three novel missense mutations (p.G31S, p.E271K, and p.I286T) and a recently described mutation (p.R326Q) were identified. The p.R326Q mutation was detected in all 13 patients with eight patients being homozygous for this mutation. Screening for the p.R326Q mutation in 110 Japanese individuals showed an allele frequency of 0.9 %. Transient expression of mutant beta UP enzymes in HEK293 cells showed that the p.E271K and p.R326Q mutations cause profound decreases in activity (a parts per thousand currency sign 1.3 %). Conversely, beta UP enzymes containing the p.G31S and p.I286T mutations possess residual activities of 50 and 70 %, respectively, suggesting we cannot exclude the presence of additional mutations in the non-coding region of the UPB1 gene. Analysis of a human beta UP homology model revealed that the effects of the mutations (p.G31S, p.E271K, and p.R326Q) on enzyme activity are most likely linked to improper oligomer assembly. Highly variable phenotypes ranging from neurological involvement (including convulsions and autism) to asymptomatic, were observed in diagnosed patients. High prevalence of p.R326Q in the normal Japanese population indicates that beta UP deficiency is not as rare as generally considered and screening for beta UP deficiency should be included in diagnosis of patients with unexplained neurological abnormalities.

  • 39. Nakajima, Yoko
    et al.
    Meijer, Judith
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ito, Tetsuya
    Zhang, Chunhua
    Wang, Xu
    Watanabe, Yoriko
    Tashiro, Kyoko
    Meinsma, Rutger
    Roelofsen, Jeroen
    Zoetekouw, Lida
    van Kuilenburg, André B P
    Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity2017In: Molecular Genetics and Metabolism, ISSN 1096-7192, E-ISSN 1096-7206, Vol. 122, no 4, p. 216-222Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyzes the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 31 genetically confirmed patients with a DHP deficiency have been reported and the clinical, biochemical and genetic spectrum of DHP deficient patients is, therefore, still largely unknown. Here, we show that 4 newly identified DHP deficient patients presented with strongly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in urine and a highly variable clinical presentation, ranging from asymptomatic to infantile spasm and reduced white matter and brain atrophy. Analysis of the DHP gene (DPYS) showed the presence of 8 variants including 4 novel/rare missense variants and one novel deletion. Functional analysis of recombinantly expressed DHP mutants carrying the p.M250I, p.H295R, p.Q334R, p.T418I and the p.R490H variant showed residual DHP activities of 2.0%, 9.8%, 9.7%, 64% and 0.3%, respectively. The crystal structure of human DHP indicated that all point mutations were likely to cause rearrangements of loops shaping the active site, primarily affecting substrate binding and stability of the enzyme. The observation that the identified mutations were more prevalent in East Asians and the Japanese population indicates that DHP deficiency may be more common than anticipated in these ethnic groups.

  • 40.
    Nakajima, Yoko
    et al.
    Fujita Hlth Univ, Sch Med, Dept Pediat, Toyoake, Aichi 4701192, Japan.
    Meijer, Judith
    Univ Amsterdam, Acad Med Ctr, Lab Genet Metab Dis, Meibergdreef 9, NL-1105 AZ Amsterdam, Netherlands.
    Zhang, Chunhua
    MILS Int, Dept Res & Dev, Kanazawa, Ishikawa 9218105, Japan.
    Wang, Xu
    Capital Univ Med Sci, Beijing Childrens Hosp, Dept Neurol, Beijing 100045, Peoples R China.
    Kondo, Tomomi
    Fujita Hlth Univ, Sch Med, Dept Pediat, Toyoake, Aichi 4701192, Japan.
    Ito, Tetsuya
    Fujita Hlth Univ, Sch Med, Dept Pediat, Toyoake, Aichi 4701192, Japan.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Van Kuilenburg, André B P
    Univ Amsterdam, Acad Med Ctr, Lab Genet Metab Dis, Meibergdreef 9, NL-1105 AZ Amsterdam, Netherlands.
    Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.2016In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 17, no 1, article id 86Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  • 41.
    Porrmann, Joseph
    et al.
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Betcheva-Krajcir, Elitza
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Di Donato, Nataliya
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Kahlert, Anne-Karin
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Schallner, Jens
    Univ Klinikum Carl Gustav Carus, Childrens Hosp, Dresden, Germany..
    Rump, Andreas
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Schroeck, Evelin
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Roelofsen, Jeroen
    Univ Amsterdam, Dept Clin Chem, Lab Genet Metab Dis, Acad Med Ctr,Emma Childrens Hosp, Amsterdam, Netherlands..
    van Kuilenburg, Andre B. P.
    Univ Amsterdam, Dept Clin Chem, Lab Genet Metab Dis, Acad Med Ctr,Emma Childrens Hosp, Amsterdam, Netherlands..
    Tzschach, Andreas
    Tech Univ Dresden, Inst Klin Genet, Dresden, Germany..
    Novel PRPS1 gain-of-function mutation in a patient with congenital hyperuricemia and facial anomalies2017In: American Journal of Medical Genetics. Part A, ISSN 1552-4825, E-ISSN 1552-4833, Vol. 173, no 10, p. 2736-2742Article in journal (Refereed)
    Abstract [en]

    Phosphoribosylpyrophosphate synthetase (PRPPS) superactivity (OMIM 300661) is a rare inborn error of purine metabolism that is caused by gain-of-function mutations in the X-chromosomal gene PRPS1 (Xq22.3). Clinical characteristics include congenital hyperuricemia and hyperuricosuria, gouty arthritis, urolithiasis, developmental delay, hypotonia, recurrent infections, short stature, and hearing loss. Only eight families with PRPPS superactivity and PRPS1 gain-of-function mutations have been reported to date. We report on a 7-year-old boy with congenital hyperuricemia, urolithiasis, developmental delay, short stature, hypospadias, and facial dysmorphisms. His mother also suffered from hyperuricemia that was diagnosed at age 13 years. A novel PRPS1 missense mutation (c.573G>C, p.[Leu191Phe]) was detected in the proband and his mother. Enzyme activity analysis confirmed superactivity of PRPP synthetase. Analysis of the crystal structure of human PRPPS suggests that the Leu191Phe mutation affects the architecture of both allosteric sites, thereby preventing the allosteric inhibition of the enzyme. The family reported here broadens the clinical spectrum of PRPPS superactivity and indicates that this rare metabolic disorder might be associated with a recognizable facial gestalt.

  • 42.
    Raposo, Bruno
    et al.
    Karolinska Institutet.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ge, Changrong
    Karolinska Institutet.
    Ekman, Diana
    Karolinska Institutet.
    Xu, Bingze
    Karolinska Institutet.
    Lindh, Ingrid
    Karolinska Institutet.
    Förster, Michael
    Karolinska Institutet.
    Uysal, Huseyin
    Karolinska Institutet.
    Nandakumar, Kutty Selva
    Karolinska Institutet.
    Schneider, Gunter
    Karolinska Institutet.
    Holmdahl, Rikard
    Karolinska Institutet.
    Epitope-specific antibody response is controlled by immunoglobuline VH polymorphisms2014In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 211, no 3, p. 405-411Article in journal (Refereed)
  • 43. Schnackerz, K D
    et al.
    Andersen, G
    Dobritzsch, Doreen
    Karolinska Institutet.
    Piskur, J
    Degradation of pyrimidines in Saccharomyces kluyveri: transamination of beta-alanine2008In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 27, no 6, p. 794-799Article in journal (Refereed)
    Abstract [en]

    Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme beta -alanine aminotransferase ((Sk)Pyd4p), highly homologous to eukaryotic gamma-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase ((Sk)Uga1p) with 80% and (Sk)Pyd4p with 55% identity to S. cerevisiae GABA-AT. (Sk)Pyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for alpha-ketoglutarate (alpha KG), beta-alanine (BAL) and gamma-aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. (Sk)Uga1p accepts only alpha KG and GABA but not BAL, thus only (Sk)Pydy4p belongs to the uracil degradative pathway.

  • 44. Schnackerz, Klaus D
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Amidohydrolases of the reductive pyrimidine catabolic pathway: purification, characterization, structure, reaction mechanisms and enzyme deficiency2008In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1784, no 3, p. 431-444Article, review/survey (Refereed)
    Abstract [en]

    In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.

  • 45. Schnackerz, Klaus D
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Lindqvist, Ylva
    Cook, Paul F
    Dihydropyrimidine dehydrogenase: a flavoprotein with four iron-sulfur clusters2004In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1701, no 1-2, p. 61-74Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the pathway for degradation of pyrimidines, responsible for the reduction of the 5,6-double bond to give the dihydropyrimidine using NADPH as the reductant. The enzyme is a dimer of 220 kDa, and each monomer contains one FAD, one FMN, and four FeS clusters. The FAD is situated at one end of the protein, the FMN is at the other, and four FeS clusters form a conduit for electron transfer between the two sites comprised of two FeS clusters from each monomer. The enzyme has a two-site ping-pong mechanism with NADPH reducing FAD and reduced FMN responsible for reducing the pyrimidine. Solvent deuterium kinetic isotope effects indicate a rate-limiting reduction of FAD accompanied by pH-dependent structural rearrangement for proper orientation of the nicotinamide ring. Transfer of electrons from site 1 to site 2 is downhill with FMN rapidly reduced by FADH(2) via the FeS conduit. The reduction of the pyrimidine at site 2 proceeds using general acid catalysis with protonation at N5 of FMN carried out by K574 as FMN is reduced and protonation at C5 of the pyrimidine by C671 as it is reduced. Kinetic isotope effects indicate a stepwise reaction for reduction of the pyrimidine with hydride transfer at C6 preceding proton transfer at C5, with a late transition state for the proton transfer step.

  • 46. van Kuilenburg, A B P
    et al.
    Meijer, J
    Dobritzsch, Doreen
    Karolinska Institutet.
    Lohkamp, B
    Ruitenbeek, W
    Roelofsen, J
    Abeling, N G G M
    Duran, M
    Buzing, C
    Identification of two novel mutations C79X and R235Q in the dihydropyrimidine dehydrogenase gene in a patient presenting with hematuria2008In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 27, no 6-7, p. 809-815Article in journal (Refereed)
    Abstract [en]

    A patient with hematuria was shown to have thymine-uraciluria. The dihydropyrimidine dehydrogenase (DPD) activity in peripheral blood mononuclear cells was 0.16 nmol/mg/h; controls: 9.9 +/- 2.8 nmol/mg/h. Analysis of DPYD showed that the patient was compound heterozygous for the novel mutations 237C > A (C79X) in exon 4 and 704G > A (R235Q) in exon 7. The nonsense mutation (C79X) leads to premature termination of translation and thus to a non-functional protein. Analysis of the crystal structure of pig DPD suggested that the R235Q mutation might interfere with the binding of FAD and the electron flow between the NADPH and the pyrimidine substrate site of DPD.

  • 47.
    van Kuilenburg, Andre B. P.
    et al.
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,United Metab Dis, Amsterdam, Netherlands.
    Tarailo-Graovac, Maja
    Univ Calgary, Cumming Sch Med, Dept Biochem & Mol Biol, Calgary, AB, Canada;Univ Calgary, Cumming Sch Med, Dept Med Genet, Calgary, AB, Canada;Univ Calgary, Alberta Childrens Hosp, Res Inst, Calgary, AB, Canada.
    Richmond, Phillip A.
    Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada.
    Drogemoller, Britt I.
    Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC, Canada.
    Pouladi, Mahmoud A.
    Natl Univ Singapore, Dept Med, Singapore, Singapore;Natl Univ Singapore, Dept Physiol, Singapore, Singapore;Agcy Sci Technol & Res, Translat Lab Genet Med, Singapore, Singapore.
    Leen, Rene
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    Brand-Arzamendi, Koroboshka
    St Michaels Hosp, Zebrafish Ctr Adv Drug Discovery, Toronto, ON, Canada;Univ Toronto, Toronto, ON, Canada.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dolzhenko, Egor
    Illumina, San Diego, CA USA.
    Eberle, Michael A.
    Illumina, San Diego, CA USA.
    Hayward, Bruce
    NIDDK, Gene Struct & Dis Sect, Lab Cell & Mol Biol, NIH, Bethesda, MD 20892 USA.
    Jones, Meaghan J.
    Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada.
    Karbassi, Farhad
    St Michaels Hosp, Zebrafish Ctr Adv Drug Discovery, Toronto, ON, Canada;Univ Toronto, Toronto, ON, Canada.
    Kobor, Michael S.
    Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada.
    Koster, Janet
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    Kumari, Daman
    NIDDK, Gene Struct & Dis Sect, Lab Cell & Mol Biol, NIH, Bethesda, MD 20892 USA.
    Li, Meng
    St Michaels Hosp, Zebrafish Ctr Adv Drug Discovery, Toronto, ON, Canada;Univ Toronto, Toronto, ON, Canada.
    MacIsaac, Julia
    Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada.
    McDonald, Cassandra
    Univ British Columbia, Dept Med Genet, Vancouver, BC, Canada.
    Meijer, Judith
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    Nguyen, Charlotte
    Univ Toronto, Hosp Sick Children, Ctr Appl Genom Genet & Genome Biol, Toronto, ON, Canada;Univ Toronto, Dept Mol Genet, Toronto, ON, Canada.
    Rajan-Babu, Indhu-Shree
    Univ British Columbia, Dept Med Genet, Vancouver, BC, Canada.
    Scherer, Stephen W.
    Univ Toronto, Hosp Sick Children, Ctr Appl Genom Genet & Genome Biol, Toronto, ON, Canada;Univ Toronto, Dept Mol Genet, Toronto, ON, Canada;Univ Toronto, McLaughlin Ctr, Toronto, ON, Canada.
    Sim, Bernice
    Agcy Sci Technol & Res, Translat Lab Genet Med, Singapore, Singapore.
    Trost, Brett
    Univ Toronto, Hosp Sick Children, Ctr Appl Genom Genet & Genome Biol, Toronto, ON, Canada.
    Tseng, Laura A.
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    Turkenburg, Marjolein
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    van Vugt, Joke J. F. A.
    Univ Med Ctr Utrecht, Brain Ctr Rudolf Magnus, Dept Neurol, Utrecht, Netherlands;Project MinE ALS Sequencing Consortium, Utrecht, Netherlands.
    Veldink, Jan H.
    Univ Med Ctr Utrecht, Brain Ctr Rudolf Magnus, Dept Neurol, Utrecht, Netherlands;Project MinE ALS Sequencing Consortium, Utrecht, Netherlands.
    Walia, Jagdeep S.
    Univ Ottawa, Childrens Hosp Eastern Ontario, Dept Pediat, Div Med Genet, Ottawa, ON, Canada.
    Wang, Youdong
    St Michaels Hosp, Zebrafish Ctr Adv Drug Discovery, Toronto, ON, Canada;Univ Toronto, Toronto, ON, Canada.
    van Weeghel, Michel
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands.
    Wright, Galen E. B.
    Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC, Canada.
    Xu, Xiaohong
    Agcy Sci Technol & Res, Translat Lab Genet Med, Singapore, Singapore.
    Yuen, Ryan K. C.
    Univ Toronto, Hosp Sick Children, Ctr Appl Genom Genet & Genome Biol, Toronto, ON, Canada;Univ Toronto, Dept Mol Genet, Toronto, ON, Canada.
    Zhang, Jinqiu
    Agcy Sci Technol & Res, Translat Lab Genet Med, Singapore, Singapore.
    Ross, Colin J.
    Univ British Columbia, Fac Pharmaceut Sci, Vancouver, BC, Canada.
    Wasserman, Wyeth W.
    Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada;Univ British Columbia, Dept Med Genet, Vancouver, BC, Canada.
    Geraghty, Michael T.
    Univ Ottawa, Childrens Hosp Eastern Ontario, Dept Pediat, Div Med Genet, Ottawa, ON, Canada.
    Santra, Saikat
    Birmingham Childrens Hosp, Dept Clin Inherited Metab Disorders, Birmingham, W Midlands, England.
    Wanders, Ronald J. A.
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,United Metab Dis, Amsterdam, Netherlands.
    Wen, Xiao-Yan
    St Michaels Hosp, Zebrafish Ctr Adv Drug Discovery, Toronto, ON, Canada;Univ Toronto, Toronto, ON, Canada;Univ Toronto, Inst Med Sci, Dept Med, Toronto, ON, Canada;Univ Toronto, Inst Med Sci, Dept Physiol, Toronto, ON, Canada;Univ Toronto, Inst Med Sci, Dept Lab Med & Pathobiol, Toronto, ON, Canada.
    Waterham, Hans R.
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,United Metab Dis, Amsterdam, Netherlands.
    Usdin, Karen
    NIDDK, Gene Struct & Dis Sect, Lab Cell & Mol Biol, NIH, Bethesda, MD 20892 USA.
    van Karnebeek, Clara D. M.
    Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,Amsterdam Gastroenterol & Metab, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Chem,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Pediat,United Metab Dis, Amsterdam, Netherlands;Univ Amsterdam, Amsterdam Univ Med Ctr, Emma Childrens Hosp, Dept Clin Genet,United Metab Dis, Amsterdam, Netherlands;Univ British Columbia, BC Childrens Hosp, Res Inst, Ctr Mol Med & Therapeut, Vancouver, BC, Canada;Univ British Columbia, Dept Pediat, Vancouver, BC, Canada.
    Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS2019In: New England Journal of Medicine, ISSN 0028-4793, E-ISSN 1533-4406, Vol. 380, no 15, p. 1433-1441Article in journal (Refereed)
    Abstract [en]

    We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5′ untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

  • 48. van Kuilenburg, André B P
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Meijer, Judith
    Krumpel, Michael
    Selim, Laila A
    Rashed, Mohamed S
    Assmann, Birgit
    Meinsma, Rutger
    Lohkamp, Bernhard
    Ito, Tetsuya
    Abeling, Nico G G M
    Saito, Kayoko
    Eto, Kaoru
    Smitka, Martin
    Engvall, Martin
    Zhang, Chunhua
    Xu, Wang
    Zoetekouw, Lida
    Hennekam, Raoul C M
    ß-ureidopropionase deficiency: phenotype, genotype and protein structural consequences in 16 patients2012In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1822, no 7, p. 1096-108Article in journal (Refereed)
    Abstract [en]

    ß-ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO(2). To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.

  • 49. van Kuilenburg, André B P
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Meijer, Judith
    Meinsma, Rutger
    Benoist, Jean-François
    Assmann, Birgit
    Schubert, Susanne
    Hoffmann, Georg F
    Duran, Marinus
    de Vries, Maaike C
    Kurlemann, Gerd
    Eyskens, François J M
    Greed, Lawrence
    Sass, Jörn Oliver
    Schwab, K Otfried
    Sewell, Adrian C
    Walter, John
    Hahn, Andreas
    Zoetekouw, Lida
    Ribes, Antonia
    Lind, Suzanne
    Hennekam, Raoul C M
    Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients2010In: Biochimica et Biophysica Acta - Molecular Basis of Disease, ISSN 0925-4439, E-ISSN 1879-260X, Vol. 1802, no 7-8, p. 639-648Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidinase (DHP) is the second enzyme of the pyrimidine degradation pathway and catalyses the ring opening of 5,6-dihydrouracil and 5,6-dihydrothymine. To date, only 11 individuals have been reported suffering from a complete DHP deficiency. Here, we report on the clinical, biochemical and molecular findings of 17 newly identified DHP deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological and gastrointestinal abnormalities and markedly elevated levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. Analysis of DPYS, encoding DHP, showed nine missense mutations, two nonsense mutations, two deletions and one splice-site mutation. Seventy-one percent of the mutations were located at exons 5-8, representing 41% of the coding sequence. Heterologous expression of 11 mutant enzymes in Escherichia coli showed that all but two missense mutations yielded mutant DHP proteins without significant activity. Only DHP enzymes containing the mutations p.R302Q and p.T343A possessed a residual activity of 3.9% and 49%, respectively. The crystal structure of human DHP indicated that the point mutations p.R490C, p.R302Q and p.V364M affect the oligomerization of the enzyme. In contrast, p.M70T, p.D81G, p.L337P and p.T343A affect regions near the di-zinc centre and the substrate binding site. The p.S379R and p.L7V mutations were likely to cause structural destabilization and protein misfolding. Four mutations were identified in multiple unrelated DHP patients, indicating that DHP deficiency may be more common than anticipated.

  • 50. van Kuilenburg, André B P
    et al.
    Dobritzsch, Doreen
    Karolinska Institutet.
    Meinsma, Rutger
    Haasjes, Janet
    Waterham, Hans R
    Nowaczyk, Malgorzata J M
    Maropoulos, George D
    Hein, Guido
    Kalhoff, Hermann
    Kirk, Jean M
    Baaske, Holger
    Aukett, Anne
    Duley, John A
    Ward, Kate P
    Lindqvist, Ylva
    van Gennip, Albert H
    Novel disease-causing mutations in the dihydropyrimidine dehydrogenase gene interpreted by analysis of the three-dimensional protein structure2002In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 364, no Pt 1, p. 157-163Article in journal (Refereed)
    Abstract [en]

    Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria in homozygous deficient patients. Cancer patients with a partial deficiency of DPD are at risk of developing severe life-threatening toxicities after the administration of 5-fluorouracil. Thus, identification of novel disease-causing mutations is of the utmost importance to allow screening of patients at risk. In eight patients presenting with a complete DPD deficiency, a considerable variation in the clinical presentation was noted. Whereas motor retardation was observed in all patients, no patients presented with convulsive disorders. In this group of patients, nine novel mutations were identified including one deletion of two nucleotides [1039-1042delTG] and eight missense mutations. Analysis of the crystal structure of pig DPD suggested that five out of eight amino acid exchanges present in these patients with a complete DPD deficiency, Pro86Leu, Ser201Arg, Ser492Leu, Asp949Val and His978Arg, interfered directly or indirectly with cofactor binding or electron transport. Furthermore, the mutations Ile560Ser and Tyr211Cys most likely affected the structural integrity of the DPD protein. Only the effect of the Ile370Val and a previously identified Cys29Arg mutation could not be readily explained by analysis of the three-dimensional structure of the DPD enzyme, suggesting that at least the latter might be a common polymorphism. Our data demonstrate for the first time the possible consequences of missense mutations in the DPD gene on the function and stability of the DPD enzyme.

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