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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 Å 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.