Elizabeth Noey - Origins of stereoselectivity in evolved ketoreductases

Document created by Elizabeth Noey on Jan 11, 2018
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  Publication Details (including relevant citation   information):

  Noey, Elizabeth L., Tibrewal, Nidhi, Jiménez-Osés, Gonzalo,   Osuna, Sílvia, Park, Jiyong, Bond, Carly M., Cascio, Duilio,   Liang, Jack, Zhang, Xiyun, Huisman, Gjalt W., Tang, Yi, Houk,   Kendall N. 2015 112 (51 ) E7065 -E7072

  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.

  Address (URL): http://www.pnas.org/content/112/51/E7065.abstract