HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Supplemental Research Data All Versions of this Article: ps.035089.108v1 17/8/1412    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Zhang, R. Articles by Rao, Z. PubMed PubMed Citation Articles by Zhang, R. Articles by Rao, Z. Social Bookmarking                   What's this?

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

¹ Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China
² National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China
³ Crystallography Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA
⁴ Laboratory of Structural Biology, Tsinghua University, Beijing 100084, People's Republic of China
⁵ College of Life Sciences, Nankai University, Tianjin 300071, People's Republic of China

(RECEIVED February 23, 2008; FINAL REVISION April 15, 2008; ACCEPTED April 21, 2008)

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 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.

Keywords: alcohol dehydrogenase; Candida parapsilosis; short-chain dehydrogenases/reductases (SDR); X-ray crystallography; site-directed mutagenesis

CiteULike

Connotea

Del.icio.us

Digg

Reddit

Technorati