Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1633 Structure Function Relationships in Pyrimidine Degrading and Biocatalytic Enzymes, and Their Implications for Cancer Therapy and Green Chemistry DIRK MAURER ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0240-9 UPPSALA urn:nbn:se:uu:diva-341639 2018 Dissertation presented at Uppsala University to be publicly examined in B41, Husargatan 3, Uppsala, Sweden, Friday, 6 April 2018 at 09:30 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Rikkert Wierenga (University of Oulu). Abstract Maurer, D. 2018. Structure Function Relationships in Pyrimidine Degrading and Biocatalytic Enzymes, and Their Implications for Cancer Therapy and Green Chemistry. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1633. 129 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0240-9. 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. Keywords: β-Ureidopropionase, Dihydropyrimidine Dehydrogenase, Alcohol Dehydrogenase, Pyrimidine catabolism, 5-Fluorouracil, CASTing, X-ray Crystallography Dirk Maurer, Department of Chemistry - BMC, Biochemistry, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Dirk Maurer 2018 ISSN 1651-6214 ISBN 978-91-513-0240-9 urn:nbn:se:uu:diva-341639 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-341639) Für mein Schatz List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Maurer, D., Lohkamp, B., Krumpel, M., Widersten, M., Dobritzsch, D. (2018) Characterization and structure determination of human β- ureidopropionase reveal pH-dependent regulation by ligand-induced changes in oligomerization. Manuscript II van Kuilenburg, A.B.P., Meijer, J., Maurer, D., Dobritzsch, D., Meinsma, R., Los, M., Knegt, L.C., Zoetekouw, L., Jansen, R.L.H., Dezentjé, V., van Huis- Tanja, L.H., van Kampen, R.J.W., Hertz, J.M., Hennekam, R.C.M. (2017) Severe fluoropyrimidine toxicity due to novel and rare DPYD missense mu- tations, deletion and genomic amplification affecting DPD activity and mRNA splicing. Biochimica et Biophysica Acta, 1863:721-730 III van Kuilenburg, A.B.P., Tarailo-Graovac, M., Meijer, J., Drogemoller, B., Vockley, J., Maurer, D., Dobritzsch, D., Ross, C.J., Wasserman, W., Meins- ma, R., Zoetekouw, L., van Karnebeek, C.D.M. (2018) Genome sequencing reveals a novel genetic mechanism underlying dihydropyrimidine dehydro- genase deficiency: a large intragenic inversion in DPYD spanning intron 8 to intron 12. Submitted IV Hamnevik, E., Enugala, T.R., Maurer, D., Ntuku, S., Oliveira, A., Do- britzsch, D., Widersten, M. (2017) Relaxation of nonproductive binding and increased rate of coenzyme release in an alcohol dehydrogenase increases turnover with a nonpreferred alcohol enantiomer. FEBS J., 284(22):3895- 3914. V 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 VI Maurer, D., Enugala, T.R, Hamnevik, E., Bauer, P., Lüking, M., Hillier, H., Kamerlin, S.C.L., Dobritzsch, D.,Widersten, M. (2018) Stereoselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corre- sponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydro- genases. Submitted Reprints were made with permission from the respective publishers. Contribution report My Contributions to the here presented studies were as follows: Paper I Performed all experimental work and contributed to the writ- ing process. Paper II Performed the crystal structure analysis and contributed to the writing process. Paper III Performed the crystal structure analysis and contributed to the writing process. Paper IV Performed the protein crystallization studies and solved the structure of the enzyme variants. Contributed to the writing process. Paper V Performed the protein crystallization studies and solved the structure of the enzyme variants. Contributed to the writing process. Paper VI Purified the ADH-A B1F4 variant, performed the protein crystallization studies and solved the structure of both pro- duced enzyme variants. Contributed to the writing process. Contents 1 Introduction .................................................................................................... 11 2 Theoretical background .................................................................................. 13 2.1 Nucleotides and proteins – molecules of life ............................................. 13 2.1.1 DNA & RNA – the code of life ....................................................... 13 2.1.1.1 The central dogma of molecular biology............................. 14 2.1.2 Proteins – workhorses in our cells ................................................... 15 2.2 Enzymes – why study them? ..................................................................... 16 2.2.1 Enzyme kinetics ............................................................................... 18 2.2.1.1 Steady state kinetics ............................................................ 19 2.2.1.2 Pre-steady state kinetics ...................................................... 22 2.2.2 Regulation of enzyme activity ......................................................... 23 2.2.3 Enzymes as drug targets .................................................................. 26 2.2.4 Enzymes in industrial applications – Green Chemistry ................... 26 2.3 X-ray crystallography ................................................................................ 27 2.3.1 From crystal to protein structure ...................................................... 27 2.3.2 Limitations of X-ray crystallography .............................................. 32 2.3.3 Molecular dynamics simulations and docking calculations ............. 33 2.3.4 Other protein structure solving methods .......................................... 34 2.4 Protein engineering .................................................................................... 35 2.4.1 Directed evolution ........................................................................... 35 2.4.2 Rational design ................................................................................ 35 2.4.3 Semi-rational design ........................................................................ 36 2.5 The reductive pyrimidine degradation pathway ......................................... 37 2.5.1 Dihydropyrimidine dehydrogenase ................................................. 38 2.5.1.1 Dihyrdopyrimidine dehydrogenase