Journal
COMPUTATIONAL BIOLOGY AND CHEMISTRY
Volume 70, Issue -, Pages 89-95Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.compbiolchem.2017.08.004
Keywords
7 alpha-Hydroxysteroid dehydrogenase; Clostridium absonum; Coenzyme specificity; Enzyme engineering; Molecular dynamics simulation
Funding
- Fundamental Research Funds for the Central Universities [106112016CDJCR231213, 106112015CDJXY230001]
- National Science and Technology Major Projects for Major New Drugs Innovation and Development [2014ZX09301306-007]
- Natural Science Foundation Project of CQ CSTC [cstc2015jcyjA10094]
- Three Gorges Natural Medicine Engineering Research Center of Chongqing University of Education [167011]
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Studies of the molecular determinants of coenzyme specificity help to reveal the structure-function relationship of enzymes, especially with regards to coenzyme specificity-determining sites (CSDSs) that usually mediate complex interactions. NADP(H)-dependent 7 alpha-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7 alpha-HSDH), a member of the short-chain dehydrogenase/reductase superfamily (SDRs), possesses positively charged CSDSs that mainly contain T15, R16, R38, and R194, forming complicated polar interactions with the adenosine ribose C2 phosphate group of NADP(H). The R38 residue is crucial for coenzyme anchoring, but the influence of the other residues on coenzyme utilization is still not clear. Hence, we performed alanine scanning mutagenesis and molecular dynamic (MD) simulations. The results suggest that the natural CSDSs have the greatest NADP(H)-binding affinity, but not the best activity (k(cat)) toward NADP(+). Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (k(cat)/K-m), which was more than three-times that of the wild type. MD simulation and kinetics analysis suggested that the importance of the CSDSs of CA 7 alpha-HSDH should be in accordance with the following order R38 > T15 > R16 > R194, and S39 may have a supporting role in NADP(H) anchoring for mutants R16A/T194A and T15A/R16A/T194A. (C) 2017 Published by Elsevier Ltd.
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