Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1318 Aldolases for Enzymatic Carboligation Directed Evolution and Enzyme Structure-Function Relationship Studies HUAN MA ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-554-9411-7 UPPSALA urn:nbn:se:uu:diva-266902 2015 Dissertation presented at Uppsala University to be publicly examined in B22, BMC, Husargatan3, Uppsala, Friday, 5 February 2016 at 13:15 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Prof. Dr. Wolf- Dieter Fessner (Organische Chemie, Technische Universität Darmstadt ). Abstract Ma, H. 2015. Aldolases for Enzymatic Carboligation. Directed Evolution and Enzyme Structure-Function Relationship Studies. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1318. 67 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-554-9411-7. The research summarized in this thesis focuses on directed evolution and enzyme mechanism studies of two aldolases: 2-deoxyribose-5-phosphate aldolase (DERA) and fructose-6- phosphate aldolase (FSA). Aldolases are nature’s own catalysts for one of the most fundamental reactions in organic chemistry: the formation of new carbon-carbon bonds. In biological systems, aldol formation and cleavage reactions play central roles in sugar metabolism. In organic synthesis, aldolases attract great attention as environmentally friendly alternative for the synthesis of polyhydroxylated compounds in stereocontrolled manner. However, naturally occurring aldolases can hardly be used directly in organic synthesis mainly due to their narrow substrate scopes, especially phosphate dependency on substrate level. Semi-rational directed evolution was used in order to investigate the possibility of expanding the substrate scope of both DERA and FSA and to understand more about the relationship between protein structure and catalytic properties. The first two projects focus on the directed evolution of DERA and studies of the enzyme mechanism. The directed evolution project aims to alter the acceptor substrate preference from phosphorylated aldehydes to aryl-substituted aldehydes. Effort has been made to develop screening methods and screen for variants with desired properties. In the study of enzyme mechanism where enzyme steady state kinetic studies were combined with molecular dynamic simulations, we investigated the role of Ser238 and Ser239 in the phosphate binding site and the possible connection between enzyme dynamics and catalytic properties. The other two projects focus on the directed evolution of FSA and the development of a new screening assay facilitating screening for FSA variants with improved activity in catalyzing aldol reaction between phenylacetaldehyde and hydroxyacetone. The new assay is based on a coupled enzyme system using an engineered alcohol dehydrogenase, FucO DA1472, as reporting enzyme. The assay has been successfully used to identify a hit with 9-fold improvement in catalytic efficiency and to determine the steady state kinetic parameters of wild-type FSA as well as the mutants. The results from directed evolution illustrated the high degree malleability of FSA active site. This opens up possibilities to generate FSA variants which could utilize both aryl-substituted donor and acceptor substrates. Keywords: aldolase, 2-deoxyribose-5-phosphate aldolase, fructose-6-phosphate aldolase, directed evolution, enzyme dynamics Huan Ma, Department of Chemistry - BMC, Biochemistry, Box 576, Uppsala University, SE-75123 Uppsala, Sweden. © Huan Ma 2015 ISSN 1651-6214 ISBN 978-91-554-9411-7 urn:nbn:se:uu:diva-266902 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-266902) List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Ma, H., Szeler, K., Kamerlin, S. C. L. and Widersten, M. (2015) Linking Coupled Motions and Entropic Effects to the Catalytic Ac- tivity of 2-deoxyribose-5-phosphate Aldolase (DERA). Chem. Sci., in the press, DOI: 10.1039/C5SC03666F. II Ma, H., Enugala, T. R. and Widersten, M. (2015) A Microplate Format Assay for Real-Time Screening for New Aldolases that Ac- cept Aryl-Substituted Acceptor Substrate. ChemBioChem, in the press, DOI: 10.1002/cbic.201500466. Ma, H., Enugala, T. R. and Widersten, M. (2015) Directed Evolution of Fructose-6-Phosphate Aldolase Towards Improved Activity with Aryl-Substituted Acceptor Substrates. Manuscript. Reprints were made with permission from the respective publishers. Publication not included in this thesis IV Janfalk Carlsson, Å., Bauer, P., Ma, H. and Widersten, M. (2012) Obtaining Optical Purity for Product Diols in Enzyme-Catalyzed Epoxide Hydrolysis: Contributions from Changes in both Enantio- and Regioselectivity. Biochemistry, 51: 7627-7637. Contents Introduction ................................................................................................... 11 1. Biocatalysis and biocatalysts ................................................................ 11 2. Directed evolution ................................................................................ 13 3. Saturation mutagenesis and codon denegeracy .................................... 14 4. Molecular dynamics simulation ............................................................ 16 5. Enzyme dynamics and its potential link to enzyme catalysis ............... 17 6. Aldolases .............................................................................................. 18 6.1 Aldol reaction ................................................................................. 18 6.2 Aldolases ........................................................................................ 18 6.3 Class I aldolases ............................................................................. 19 7. Aldolases studied in this thesis ............................................................. 20 7.1 2-Deoxyribose-5-phosphate aldolase: DERA ................................ 20 7.2 Fructose-6-phosphate aldolase: FSA ............................................. 23 Present Investigations .................................................................................... 25 Present Investigation I: Directed evolution of DERA- an attempt to expand the acceptor substrate scope to non-phosphorylated aromatic compounds. ............................................................................................... 26 1. Aim .................................................................................................. 26 2. The design of enzyme libraries ........................................................ 27 3. Screening assay using thin layer chromatography and 14C labeled acetaldehyde. ........................................................................................ 28 4. Screening results and hits sequence analysis ................................... 30 5. Conclusion and discussion ............................................................... 31 Present Investigation II: Enzyme kinetic study and molecular dynamics simulation on wild-type DERA and related mutants. ............................... 33 1. Aim .................................................................................................. 33 2. Choice of enzymes ........................................................................... 33 3. Role of phosphate binding in DERA ............................................... 34 4. Enzyme steady state kinetic analysis ............................................... 35 5. Molecular dynamics simulation ....................................................... 38 6. Conclusion ....................................................................................... 40 Present Investigation III: Development of screening assay for FSA variants with improved activity towards phenylacetaldehyde .................. 41 1. Aim .................................................................................................. 41 2. Development of a coupled enzyme assay for detecting the formation of phenylacetaldehyde ......................................................................... 41 3. Steady state kinetic study of FSA in aldol cleavage reaction .......... 42 4. Test screening on 96-well plate ....................................................... 43 5. Conclusion ....................................................................................... 45 Present Investigation IV: Directed evolution of FSA ............................... 46 1. Aim .................................................................................................. 46 2. Library design .................................................................................. 46 3. Library screening and identification of hits ..................................... 47 4. Steady state kinetic studies on first generation library hits .............. 49 5. Conclusion and future plan .............................................................. 49 Concluding Remarks and Future Perspectives .............................................. 51 Populärvetenskaplig Sammanfattning ........................................................... 52 Résumé .......................................................................................................... 55 论文摘要 ....................................................................................................... 58 Acknowledgements ......................................................................................