Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity

A novel short- chain ( S)- 1- phenyl- 1,2- ethanediol dehydrogenase ( SCR) from Candida parapsilosis exhibits coenzyme specificity for NADPH over NADH. It catalyzes an anti- Prelog type reaction to reduce 2- hydroxyacetophenone into ( S)- 1- phenyl- 1,2- ethanediol. The coding gene was overexpressed in Escherichia coli and the purified protein was crystallized. The crystal structure of the apo- form was solved to 2.7 A resolution. This protein forms a homo- tetramer with a broken 2- 2- 2 symmetry. The overall fold of each SCR subunit is similar to that of the known structures of other homologous alcohol dehydrogenases, although the latter usually form tetramers with perfect 2- 2- 2 symmetries. Additionally, in the apo- SCR structure, the entrance of the NADPH pocket is blocked by a surface loop. In order to understand the structure - function relationship of SCR, we carried out a number of mutagenesis enzymatic analyses based on the new structural information. First, mutations of the putative catalytic Ser- Tyr- Lys triad confirmed their functional role. Second, truncation of an N- terminal 31- residue peptide indicated its role in oligomerization, but not in catalytic activity. Similarly, a V270D point mutation rendered the SCR as a dimer, rather than a tetramer, without affecting the enzymatic activity. Moreover, the S67D/ H68D double- point mutation inside the coenzyme- binding pocket resulted in a nearly 10- fold increase and a 20- fold decrease in the kcat/ KM value when NADH and NADPH were used as cofactors, respectively, with kcat remaining essentially the same. This latter result provides a new example of a protein engineering approach to modify the coenzyme specificity in SCR and short- chain dehydrogenases/ reductases in general.