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  • 1.
    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.

  • 2.
    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.

  • 3.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University.
    Structure Function Relationships in Pyrimidine Degrading and Biocatalytic Enzymes, and Their Implications for Cancer Therapy and Green Chemistry2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis includes the work of two separate projects, studies on pyrimidine degrading enzymes and studies on in vitro evolved enzymes. The common denominator of both projects was the use of structural information to explain functional effects, observed in the studied biocatalysts.

    In humans, and other eukaryotic organisms, the nucleobases uracil and thymine are catabolized by the reductive pyrimidine degradation pathway. This pathway is one of the factors that control the pyrimidine nucleotide concentrations in a cell. Furthermore, it is the main clearance route for pyrimidine analogues, often used as cancer drugs, like 5-fluorouracil and other fluoropyrimidines. Deficiencies in any of the enzymes, involved in this pathway, can lead to a wide range of neurological disorders, and possibly fatal fluoropyrimidine toxicity in cancer patients. Two out of the three involved enzymes, dihydropyrimidine dehydrogenase (DPD) and β-ureidopropionase (βUP), were studied in the first project of this thesis. This resulted in the first crystal structure of a human β-ureidopropionase variant, which could be used to explain functional characteristics of the enzyme. Structural analyses on novel DPD variants, found in patients suffering from DPD deficiency, could explain the decrease in catalytic activity of these enzyme variants. This strategy, of using structural information to predict functional effects from sequential mutations, has the potential to be used as a cheap and fast first assessment of possible deficiencies in this pathway.

    Enzymes are, however, not only involved in many diseases, but also used for industrial applications. The substitution of classical organic synthetic reactions with enzyme catalyzed reactions usually has a beneficial influence on environmental pollution, as illustrated in the principles of Green Chemistry. The major drawback of the use of enzymes for these purposes is their natural selectivity towards a small group of possible substrates and products, which often do not have the desired composition or conformation for an industrial application. In order to improve an enzyme for industrial purposes, the alcohol dehydrogenase ADH-A, from Rhodococcus ruber, was subjected to a semi-rational approach of directed evolution, using iterative saturation mutagenesis (ISM), in the second project of this thesis. This resulted in different enzyme variants that showed the desired improvements in activity. Most functional improvements could be rationalized with the help of structural information and molecular dynamics simulations. This showed that artificial protein design has the potential to produce enzyme variants capable of substituting many organic synthetic reactions, and that structural information can play a key role in the designing process.

    List of papers
    1. Characterization and structure determination of human β-ureidopropionase reveal pH-dependent regulation by ligand-induced changes in oligomerization
    Open this publication in new window or tab >>Characterization and structure determination of human β-ureidopropionase reveal pH-dependent regulation by ligand-induced changes in oligomerization
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    β-alanine synthase, β-ureidopropionase, β-alanine, pyrimidine degradation, fluoropyrimidine toxicity
    National Category
    Biochemistry and Molecular Biology Structural Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-341994 (URN)
    Available from: 2018-02-18 Created: 2018-02-18 Last updated: 2018-02-21
    2. Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing
    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
    Show others...
    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
    3. 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
    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
    Show others...
    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
    4. Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer
    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
    Show others...
    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: 2019-10-21Bibliographically approved
    5. Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin
    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
    Show others...
    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: 2019-10-21Bibliographically approved
    6. Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
    Open this publication in new window or tab >>Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases
    Show others...
    (English)In: Article in journal (Other academic) Submitted
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-318982 (URN)
    Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-02-18
  • 4.
    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)
  • 5.
    Maurer, Dirk
    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.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lüking, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
    Hillier, Heidi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Kamerlin, Shina C. Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol DehydrogenasesIn: Article in journal (Other academic)
  • 6.
    Maurer, Dirk
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Hamnevik, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Molecular Cell Biology.
    Lüking, Malin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Petrovic, Dusan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Hillier, Heidi
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. 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, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases2018In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 8, no 8, p. 7526-7538Article in journal (Refereed)
    Abstract [en]

    ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites.

    Keywords: alcohol dehydrogenase; alcohol oxidation; biocatalysis; crystal structure; directed evolution; enzyme engineering; molecular dynamics simulations; stereoselectivity

  • 7.
    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.

  • 8. van Kuilenburg, André B P
    et al.
    Meijer, Judith
    Maurer, Dirk
    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.
    Meinsma, Rutger
    Los, Maartje
    Knegt, Lia C
    Zoetekouw, Lida
    Jansen, Rob L H
    Dezentjé, Vincent
    van Huis-Tanja, Lieke H
    van Kampen, Roel J W
    Hertz, Jens Michael
    Hennekam, Raoul C M
    Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mutations, deletion and genomic amplification affecting DPD activity and mRNA splicing2017In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1863, no 3, p. 721-730Article in journal (Refereed)
    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.

  • 9.
    van Kuilenburg, André
    et al.
    Univ Amsterdam, Emma Childrens Hosp, Lab Genet Metabol Dis Pediat & Clin Genet, Dept Clin Chem,Acad Med Ctr, Amsterdam, Netherlands.
    Tarailo-Graovac, Maja
    Univ Calgary, Cumming Sch Med, Dept Biochem, Calgary, AB, Canada; Univ Calgary, Cumming Sch Med, Dept 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.
    Meijer, Judith
    Univ Amsterdam, Emma Childrens Hosp, Lab Genet Metabol Dis Pediat & Clin Genet, Dept Clin Chem,Acad Med Ctr, Amsterdam, Netherlands.
    Drogemoller, Britt
    Univ British Columbia, Ctr Mol Med & Therapeut, Dept Pediat, Vancouver, BC, Canada; Univ British Columbia, Ctr Mol Med & Therapeut, Dept Med Genet, Vancouver, BC, Canada.
    Vockley, Gerard
    Univ Pittsburgh, Sch Med, Dept Pediat, Pittsburgh, PA 15261 USA; Univ Pittsburgh, Grad Sch Publ Hlth, Pittsburgh, PA USA.
    Maurer, Dirk
    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.
    Ross, Colin
    Univ British Columbia, Ctr Mol Med & Therapeut, Dept Pediat, Vancouver, BC, Canada; Univ British Columbia, Ctr Mol Med & Therapeut, Dept Med Genet, Vancouver, BC, Canada.
    Wasserman, Wyeth
    Univ British Columbia, Ctr Mol Med & Therapeut, Dept Pediat, Vancouver, BC, Canada; Univ British Columbia, Ctr Mol Med & Therapeut, Dept Med Genet, Vancouver, BC, Canada.
    Meinsma, Rutger
    Univ Amsterdam, Emma Childrens Hosp, Lab Genet Metabol Dis Pediat & Clin Genet, Dept Clin Chem,Acad Med Ctr, Amsterdam, Netherlands.
    Zoetekouw, Lida
    Univ Amsterdam, Emma Childrens Hosp, Lab Genet Metabol Dis Pediat & Clin Genet, Dept Clin Chem,Acad Med Ctr, Amsterdam, Netherlands.
    van Karnebeek, Clara
    Univ Amsterdam, Emma Childrens Hosp, Lab Genet Metabol Dis Pediat & Clin Genet, Dept Clin Chem,Acad Med Ctr, Amsterdam, Netherlands; Univ British Columbia, Ctr Mol Med & Therapeut, Dept Pediat, Vancouver, BC, Canada; Univ British Columbia, Ctr Mol Med & Therapeut, Dept Med Genet, Vancouver, BC, Canada.
    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 122018In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 39, no 7, p. 947-953Article in journal (Refereed)
    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.

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