Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 112, Issue 51, Pages E7065-E7072Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1507910112
Keywords
directed evolution; crystallographic structures; molecular dynamics; theozyme; enantioselectivity
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Funding
- National Institutes of Health [P41 RR015301, P41 GM103403]
- US Department of Energy [DE-AC02-06CH11357, DE-FC03-02ER63421]
- National Science Foundation [OCI-1053575]
- University of California, Los Angeles (UCLA) Institute for Digital Research and Education's Hoffman2 computer cluster
- National Institutes of Health, National Institute of General Medical Sciences [GM036700, GM097200, GM075962]
- Ministerio de Economia y Competitividad (Juan de la Cierva Postdoctoral Grant) [JCI-2012-14438]
- European Commission [PCIG14-GA-2013-630978]
- NIH, NIGMS [T32 GM067555-11]
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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. Thismethod, 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.
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