3CTM
Crystal Structure of a Carbonyl Reductase from Candida Parapsilosis with anti-Prelog Stereo-specificity
Summary for 3CTM
| Entry DOI | 10.2210/pdb3ctm/pdb |
| Related | 1H5Q |
| Descriptor | Carbonyl Reductase (3 entities in total) |
| Functional Keywords | alcohol dehydrogenase, candida parapsilosis, short-chain dehydrogenases/reductases (sdr), oxidoreductase |
| Biological source | Candida parapsilosis (Yeast) More |
| Total number of polymer chains | 8 |
| Total formula weight | 250044.29 |
| Authors | |
| Primary citation | 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 Cited by 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: 18566346DOI: 10.1110/ps.035089.108 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.69 Å) |
Structure validation
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