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

Abstract

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 mutagenesisenzymatic 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 k cat /K M value when NADH and NADPH were used as cofactors, respectively, with k cat 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.

Cyanobacterial conversion of carbon dioxide to 2,3-butanediol

“…4B). The major 23BD product of each of the adh expressing strains except the strain containing adh (C. parapsilosis) matched the stereochemistry predicted by previous characterization (38)(39)(40).…”

“…4). These results indicate that sADH (C. parapsilosis) does not show stereoselectivity toward (R)-acetoin, although this enzyme catalyzes an S-installing reaction toward 2-hydroxyacetophenone (39). We assume that (R)-acetoin is too small to bind to the catalytic site stereospecifically.…”

“…Assuming uniform diffusion this corresponds to 1.4 mM concentrations in the cell. The K m of sADH (C. parapsilosis) may be large, given that the native substrate of the enzyme has a phenyl ring adjacent to the reaction site and binding for acetoin without the phenyl ring may be inefficient (39). In this case the enzyme would have difficulty competing with diffusion of acetoin, yet would display high activity under assay conditions where acetoin concentration is high (25 mM).…”

Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

“…The structure of RasADH was solved by molecular replacement using a monomer of a 'probable dehydrogenase protein' from Rhizobium etli CFN42 (55% sequence identity with RasADH; PDB code 4FGS) as a model. In the structure solution there were four monomers A-D in the asymmetric unit, which made up a tetramer (Figure 2a), a quaternary association common to some other short-chain ADHs such as the carbonyl reductase from Candida parapsilosis (PDB code 3CTM, [15]) and levodione reductase from…”

Ser67Asp and His68Asp Substitutions in Candida parapsilosis Carbonyl Reductase Alter the Coenzyme Specificity and Enantioselectivity of Ketone Reduction

“…It is a member of the SDR family and shares more than 30% similarity with mannitol-2-dehydrogenase from Agaricus bisporus (7) and the R-specific alcohol dehydrogenase from Lactobacillus brevis (20). The crystal structure of SCR was determined recently by a molecular replacement method in our labs (28). In this work, in order to explore the possibility of converting SCR from a NADPHdependent enzyme into an NADH-dependent one, we designed mutations with different combinations of Ser67Asp, His68Asp, and Pro69Asp substitutions inside or adjacent to the so-called phosphate-binding loop between ␤B and ␣C.…”