| Autor: |
Noey EL; Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;, Tibrewal N; Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095;, Jiménez-Osés G; Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;, Osuna S; Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;, Park J; Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095;, Bond CM; Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095;, Cascio D; Molecular Biology Institute, University of California, Los Angeles, CA 90095;, Liang J; Codexis Inc., Redwood City, CA 94063., Zhang X; Codexis Inc., Redwood City, CA 94063 zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu., Huisman GW; Codexis Inc., Redwood City, CA 94063 zhangxiy@hotmail.com Gjalt.Huisman@codexis.com yitang@ucla.edu houk@chem.ucla.edu., Tang Y; 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., Houk KN; 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. |
| Abstrakt: |
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. |
