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van Kuilenburg, A. B. P., Tarailo-Graovac, M., Richmond, P. A., Drogemoller, B. I., Pouladi, M. A., Leen, R., . . . van Karnebeek, C. D. M. (2019). Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS. New England Journal of Medicine, 380(15), 1433-1441
Open this publication in new window or tab >>Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS
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2019 (English)In: New England Journal of Medicine, ISSN 0028-4793, E-ISSN 1533-4406, Vol. 380, no 15, p. 1433-1441Article in journal (Refereed) Published
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.

National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-382857 (URN)10.1056/NEJMoa1806627 (DOI)000464610000010 ()30970188 (PubMedID)
Funder
NIH (National Institute of Health), DK057808
Available from: 2019-05-15 Created: 2019-05-15 Last updated: 2019-05-15Bibliographically approved
Ge, C., Xu, B., Liang, B., Lönnblom, E., Lundström, S. L., Zubarev, R. A., . . . Holmdahl, R. (2019). Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies. Arthritis & Rheumatology, 71(2), 210-221
Open this publication in new window or tab >>Structural Basis of Cross-Reactivity of Anti-Citrullinated Protein Antibodies
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2019 (English)In: Arthritis & Rheumatology, ISSN 2326-5191, E-ISSN 2326-5205, Vol. 71, no 2, p. 210-221Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2019
National Category
Rheumatology and Autoimmunity
Identifiers
urn:nbn:se:uu:diva-377677 (URN)10.1002/art.40698 (DOI)000457458100005 ()30152126 (PubMedID)
Funder
Swedish Foundation for Strategic Research Knut and Alice Wallenberg FoundationSwedish Research CouncilEU, FP7, Seventh Framework Programme, 283570
Available from: 2019-02-26 Created: 2019-02-26 Last updated: 2019-02-26Bibliographically approved
Maurer, D., Lohkamp, B., Krumpel, M., Widersten, M. & Dobritzsch, D. (2018). Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism. Biochemical Journal, 475(14), 2395-2416
Open this publication in new window or tab >>Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism
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2018 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 475, no 14, p. 2395-2416Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-356269 (URN)10.1042/BCJ20180222 (DOI)000441396200008 ()29976570 (PubMedID)
Funder
Carl Tryggers foundation , CTS13:104Carl Tryggers foundation , CTS14:111EU, FP7, Seventh Framework Programme, 283570
Available from: 2018-07-21 Created: 2018-07-21 Last updated: 2018-10-15Bibliographically approved
Hamnevik, E., Maurer, D., Enugala, T. R., Chu, T., Löfgren, R., Dobritzsch, D. & Widersten, M. (2018). Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin. Biochemistry, 57, 1059-1062
Open this publication in new window or tab >>Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin
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2018 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, p. 1059-1062Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-340574 (URN)10.1021/acs.biochem.8b00055 (DOI)000426013300003 ()29384657 (PubMedID)
Funder
Stiftelsen Olle Engkvist Byggmästare, 183-358
Available from: 2018-01-31 Created: 2018-01-31 Last updated: 2019-06-27Bibliographically approved
Janfalk Carlsson, Å., Bauer, P., Dobritzsch, D., Kamerlin, S. C. & Widersten, M. (2018). Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme. IUCrJ, 5(3), 269-282
Open this publication in new window or tab >>Epoxide Hydrolysis as a Model System for Understanding Flux Through a Branched Reaction Scheme
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2018 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 5, no 3, p. 269-282Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-343750 (URN)10.1107/S2052252518003573 (DOI)000431151300004 ()29755743 (PubMedID)
Funder
Swedish Research CouncilEU, FP7, Seventh Framework Programme
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-12-03Bibliographically approved
van Kuilenburg, A., Tarailo-Graovac, M., Meijer, J., Drogemoller, B., Vockley, G., Maurer, D., . . . van Karnebeek, C. (2018). Genome sequencing reveals a novel genetic mechanism underlying dihydropyrimidine dehydrogenase deficiency: A novel missense variant c.1700G > A and a large intragenic inversion in DPYD spanning intron 8 to intron 12. Human Mutation, 39(7), 947-953
Open this publication in new window or tab >>Genome sequencing reveals a novel genetic mechanism underlying dihydropyrimidine dehydrogenase deficiency: A novel missense variant c.1700G > A and a large intragenic inversion in DPYD spanning intron 8 to intron 12
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2018 (English)In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 39, no 7, p. 947-953Article in journal (Refereed) Published
Abstract [en]

Dihydropyrimidine dehydrogenase (DPD) deficiency is associated with a variable clinical presentation. A family with three DPD-deficient patients presented with unusual clinical phenotypes including pregnancy-induced symptoms, transient visual impairment, severe developmental delay, cortical blindness, and delayed myelination in the brain. DPYD Sanger sequencing showed heterozygosity for the c.1905+1G>A mutation and a novel missense variant c.1700G>A (p.G567E). The recombinantly expressed p.G567E DPD variant showed increased temperature lability probably caused by structural rearrangements within the DPD protein. Genome sequencing of the affected son established compound heterozygosity for the c.1700G>A and an imperfect 115,731bp inversion with breakpoints at chr1: 98,113,121 (intron 8) and chr1: 97,997,390 (intron 12) of the DPYD associated with a 4bp deletion (chr1: 97,997,386_97,997,389del). Whole exome and mitochondrial DNA analyses for the mother and daughter did not reveal additional mutated genes of significance. Thus, an inversion in DPYD should be considered in patients with an inconclusive genotype or unusual clinical phenotype.

Keywords
dihydropyrimidine dehydrogenase, DPYD, inversion, whole genome sequencing
National Category
Medicinal Chemistry Structural Biology
Identifiers
urn:nbn:se:uu:diva-342024 (URN)10.1002/humu.23538 (DOI)000434972700006 ()29691939 (PubMedID)
Note

Title of manuscript included in thesis: Genome sequencing reveals a novel genetic mechanism underlying dihydropyrimidine dehydrogenase deficiency: a large intragenic inversion in DPYD spanning intron 8 to intron 12

Available from: 2018-02-18 Created: 2018-02-18 Last updated: 2018-08-31Bibliographically approved
Hampel, S., Steitz, J.-P., Baierl, A., Lehwald, P., Wiesli, L., Richter, M., . . . Mueller, M. (2018). Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens. ChemBioChem (Print), 19(21), 2283-2292
Open this publication in new window or tab >>Structural and Mutagenesis Studies of the Thiamine-Dependent, Ketone-Accepting YerE from Pseudomonas protegens
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 21, p. 2283-2292Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH, 2018
Keywords
asymmetric synthesis, biocatalysis, biosynthesis, C-C coupling, tertiary alcohols
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-387253 (URN)10.1002/cbic.201800325 (DOI)000449745700007 ()30101542 (PubMedID)
Funder
Swedish Research CouncilGerman Research Foundation (DFG), FOR 1296EU, European Research Council, 635595
Available from: 2019-06-26 Created: 2019-06-26 Last updated: 2019-06-26Bibliographically approved
Ge, C. P., Tong, D. R., Liang, B. T., Lönnblom, E. S., Schneider, N. K., Hagert, C. U., . . . Holmdahl, R. K. (2017). Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage. JCI INSIGHT, 2(13), Article ID e93688.
Open this publication in new window or tab >>Anti-citrullinated protein antibodies cause arthritis by cross-reactivity to joint cartilage
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2017 (English)In: JCI INSIGHT, ISSN 2379-3708, Vol. 2, no 13, article id e93688Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
AMER SOC CLINICAL INVESTIGATION INC, 2017
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-331243 (URN)10.1172/jci.insight.93688 (DOI)000405181200012 ()
Funder
Swedish Foundation for Strategic Research , RB13-015Knut and Alice Wallenberg Foundation, KAW 2010.0148Swedish Research Council, 2015-02662EU, FP7, Seventh Framework Programme, 283570
Available from: 2017-10-19 Created: 2017-10-19 Last updated: 2018-07-30
Nakajima, Y., Meijer, J., Dobritzsch, D., Ito, T., Zhang, C., Wang, X., . . . van Kuilenburg, A. B. (2017). Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity. Molecular Genetics and Metabolism, 122(4), 216-222
Open this publication in new window or tab >>Dihydropyrimidinase deficiency in four East Asian patients due to novel and rare DPYS mutations affecting protein structural integrity and catalytic activity
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2017 (English)In: Molecular Genetics and Metabolism, ISSN 1096-7192, E-ISSN 1096-7206, Vol. 122, no 4, p. 216-222Article in journal (Refereed) Published
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.

Keywords
Crystal structure, DPYS, Dihydropyrimidinase, Functional and structural protein analysis, Patients
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-338128 (URN)10.1016/j.ymgme.2017.10.003 (DOI)000418879000010 ()29054612 (PubMedID)
Available from: 2018-01-08 Created: 2018-01-08 Last updated: 2018-02-02Bibliographically approved
Porrmann, J., Betcheva-Krajcir, E., Di Donato, N., Kahlert, A.-K., Schallner, J., Rump, A., . . . Tzschach, A. (2017). Novel PRPS1 gain-of-function mutation in a patient with congenital hyperuricemia and facial anomalies. American Journal of Medical Genetics. Part A, 173(10), 2736-2742
Open this publication in new window or tab >>Novel PRPS1 gain-of-function mutation in a patient with congenital hyperuricemia and facial anomalies
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2017 (English)In: American Journal of Medical Genetics. Part A, ISSN 1552-4825, E-ISSN 1552-4833, Vol. 173, no 10, p. 2736-2742Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
WILEY, 2017
Keywords
hyperuricemia, phosphoribosylpyrophosphate synthetase superactivity, PRPS1
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-336298 (URN)10.1002/ajmg.a.38359 (DOI)000411036600023 ()28742244 (PubMedID)
Available from: 2018-01-23 Created: 2018-01-23 Last updated: 2018-01-23Bibliographically approved
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Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1822-6513

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