Related Experiment Video
Updated: Feb 24, 2026

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments
Published on: October 2, 2012
Functional contribution of coenzyme specificity-determining sites of 7α-hydroxysteroid dehydrogenase from Clostridium
Deshuai Lou1, Yue Wang2, Jun Tan3
1Postdoctoral Research Station of Biology, Chongqing University, Chongqing 400030, China; Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400030, China.
Abstract:
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 7α-hydroxysteroid dehydrogenase from Clostridium absonum (CA 7α-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 (kcat) toward NADP+. Compared with the wild type and other mutants, the mutant R194A showed the highest catalytic efficiency (kcat/Km), 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 7α-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.
Related Concept Videos
Cofactors and Coenzymes
Cofactors can be metallic ions or organic molecules called coenzymes. These types of helper...
Cofactors and Coenzymes
Ligand Binding and Linkage
Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Amino Acid Catabolism
Lipid Catabolism

