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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: 2018-07-13Bibliographically 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
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
Hamnevik, E., Enugala, T. R., Maurer, D., Ntuku, S., Oliveira, A., Dobritzsch, D. & Widersten, M. (2017). Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer. The FEBS Journal, 284(22), 3895-3914
Open this publication in new window or tab >>Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
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2017 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 284, no 22, p. 3895-3914Article in journal (Refereed) Published
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.

Keywords
alcohol dehydrogenase, biocatalysis, stereoselectivity, directed evolution, crystal structures, enzyme kinetics
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-318981 (URN)10.1111/febs.14279 (DOI)000415877100011 ()
Funder
Swedish Research Council, 621-2011-6055
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-03-09Bibliographically approved
van Kuilenburg, A. B., Meijer, J., Maurer, D., Dobritzsch, D., Meinsma, R., Los, M., . . . Hennekam, R. C. (2017). Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing. Biochimica et Biophysica Acta, 1863(3), 721-730
Open this publication in new window or tab >>Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing
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2017 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1863, no 3, p. 721-730Article in journal (Refereed) Published
Abstract [en]

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of 5-fluorouracil (5FU). Genetic variations in DPD have emerged as predictive risk factors for severe fluoropyrimidine toxicity. Here, we report novel and rare genetic variants underlying DPD deficiency in 9 cancer patients presenting with severe fluoropyrimidine-associated toxicity. All patients possessed a strongly reduced DPD activity, ranging from 9 to 53% of controls. Analysis of the DPD gene (DPYD) showed the presence of 21 variable sites including 4 novel and 4 very rare aberrations: 3 missense mutations, 2 splice-site mutations, 1 intronic mutation, a deletion of 21 nucleotides and a genomic amplification of exons 9-12. Two novel/rare variants (c.2843T>C, c.321+1G>A) were present in multiple, unrelated patients. Functional analysis of recombinantly-expressed DPD mutants carrying the p.I948T and p.G284V mutation showed residual DPD activities of 30% and 0.5%, respectively. Analysis of a DPD homology model indicated that the p.I948T and p.G284V mutations may affect electron transfer and the binding of FAD, respectively. cDNA analysis showed that the c.321+1G>A mutation in DPYD leads to skipping of exon 4 immediately upstream of the mutated splice-donor site in the process of DPD pre-mRNA splicing. A lethal toxicity in two DPD patients suggests that fluoropyrimidines combined with other therapies such as radiotherapy might be particularly toxic for DPD deficient patients. Our study advocates a more comprehensive genotyping approach combined with phenotyping strategies for upfront screening for DPD deficiency to ensure the safe administration of fluoropyrimidines.

Keywords
Dihydropyrimidine dehydrogenase, DPYD, 5-Fluorouracil, Capecitabine, Pharmacogenetics, Toxicity
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-312179 (URN)10.1016/j.bbadis.2016.12.010 (DOI)000394190300009 ()28024938 (PubMedID)
Available from: 2017-01-06 Created: 2017-01-06 Last updated: 2018-02-18
Nakajima, Y., Meijer, J., Zhang, C., Wang, X., Kondo, T., Ito, T., . . . Van Kuilenburg, A. B. (2016). Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.. International Journal of Molecular Sciences, 17(1), Article ID 86.
Open this publication in new window or tab >>Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.
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2016 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 17, no 1, article id 86Article in journal (Refereed) Published
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.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-287443 (URN)10.3390/ijms17010086 (DOI)000374583800082 ()26771602 (PubMedID)
Available from: 2016-04-25 Created: 2016-04-25 Last updated: 2018-01-10Bibliographically approved
Bauer, P., Janfalk Carlsson, Å., Amrein, B. A., Dobritzsch, D., Widersten, M. & Kamerlin, S. C. (2016). Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1. Organic and biomolecular chemistry, 14(24), 5639-5651
Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
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2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
Funder
Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
Doak, B. C., Zheng, J., Dobritzsch, D. & Kihlberg, J. (2016). How Beyond Rule of 5 Drugs and Clinical Candidates Bind to Their Targets.. Journal of Medicinal Chemistry, 59(6), 2312-2327
Open this publication in new window or tab >>How Beyond Rule of 5 Drugs and Clinical Candidates Bind to Their Targets.
2016 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 59, no 6, p. 2312-2327Article in journal (Refereed) Published
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.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-287442 (URN)10.1021/acs.jmedchem.5b01286 (DOI)000372946500003 ()26457449 (PubMedID)
Available from: 2016-04-25 Created: 2016-04-25 Last updated: 2018-07-30
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1822-6513

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