3CTM

Crystal Structure of a Carbonyl Reductase from Candida Parapsilosis with anti-Prelog Stereo-specificity

3CTM の概要

• エントリーDOI: 10.2210/pdb3ctm/pdb
• 関連するPDBエントリー: 1H5Q
• 分子名称: Carbonyl Reductase (3 entities in total)
• 機能のキーワード: alcohol dehydrogenase, candida parapsilosis, short-chain dehydrogenases/reductases (sdr), oxidoreductase
• 由来する生物種: Candida parapsilosis (Yeast); 詳細
• タンパク質・核酸の鎖数: 8
• 化学式量合計: 250044.29

構造登録者

Zhang, R.,Zhu, G.,Li, X.,Xu, Y.,Zhang, X.C.,Rao, Z. (登録日: 2008-04-14, 公開日: 2008-05-27, 最終更新日: 2024-11-20)

主引用文献

Zhang, R.,Zhu, G.,Zhang, W.,Cao, S.,Ou, X.,Li, X.,Bartlam, M.,Xu, Y.,Zhang, X.C.,Rao, Z.
Crystal structure of a carbonyl reductase from Candida parapsilosis with anti-Prelog stereospecificity.
Protein Sci., 17:1412-1423, 2008

PubMed 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 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.

PubMed: 18566346
DOI: 10.1110/ps.035089.108

実験手法

X-RAY DIFFRACTION (2.69 Å)