title: Origins of stereoselectivity in evolved ketoreductases.
abstract: Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone. X-ray crystal structures of apo- and NADP(+)-bound selected mutants show that the substrate-binding loop conformational preferences are modified by these mutations. Quantum mechanical calculations and molecular dynamics (MD) simulations are used to investigate the mechanism of reduction by the enzyme. We have developed an MD-based method for studying the diastereomeric transition state complexes and rationalize different enantiomeric ratios. This method, which probes the stability of the catalytic arrangement within the theozyme, shows a correlation between the relative fractions of catalytically competent poses for the enantiomeric reductions and the experimental enantiomeric ratio. Some mutations, such as A94F and Y190F, induce conformational changes in the active site that enlarge the small binding pocket, facilitating accommodation of the larger S atom in this region and enhancing S-selectivity with 3-thiacyclopentanone. In contrast, in the E145S mutant and the final variant evolved for large-scale production of the intermediate for the antibiotic sulopenem, R-selectivity is promoted by shrinking the small binding pocket, thereby destabilizing the pro-S orientation.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Noey
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095;
authors.forename: Nidhi
authors.initials: N
authors.lastname: Tibrewal
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Jiménez-Osés
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;
authors.forename: Sílvia
authors.initials: S
authors.lastname: Osuna
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;
authors.forename: Jiyong
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095;
authors.forename: Carly M
authors.initials: CM
authors.lastname: Bond
authors.affiliation: Molecular Biology Institute, University of California, Los Angeles, CA 90095;
authors.forename: Duilio
authors.initials: D
authors.lastname: Cascio
authors.affiliation: Codexis Inc., Redwood City, CA 94063.
authors.forename: Jack
authors.initials: J
authors.lastname: Liang
authors.affiliation: Codexis Inc., Redwood City, CA 94063 zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu.
authors.forename: Xiyun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Codexis Inc., Redwood City, CA 94063 zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu.
authors.forename: Gjalt W
authors.initials: GW
authors.lastname: Huisman
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095; zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu.
authors.forename: Yi
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095; Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095; zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu.
authors.forename: Kendall N
authors.initials: KN
authors.lastname: Houk
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011789:Quantum Theory; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases
keywords: crystallographic structures; directed evolution; enantioselectivity; molecular dynamics; theozyme