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 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.
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Rongzhen
R
Zhang
Guangyu
G
Zhu
Wenchi
W
Zhang
Sheng
S
Cao
Xianjin
X
Ou
Xuemei
X
Li
Mark
M
Bartlam
Yan
Y
Xu
Xuejun C
XC
Zhang
Zihe
Z
Rao
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D005026:Ethylene Glycols / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity
D005026:Ethylene Glycols; D005656:Fungal Proteins; D009243:NAD; D009249:NADP; D000426:Alcohol Dehydrogenase