AEM Accepts, published online ahead of print on 15 June 2007

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Appl. Environ. Microbiol. doi:10.1128/AEM.02993-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Cloning of a Novel Aldo-Keto Reductase Gene from Klebsiella sp. F51-1-2 and Its Functional Expression in Escherichia coli

Hong Jiang, Hong Qu, Chao Yang, Zheng Liu, Q. S. Fu, and Chuanling Qiao^*

State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China; Graduate School of the Chinese Academy of Sciences, Beijing 100049, China; College of Life Sciences, Peking University, Beijing 100871, China; Department of Civil and Environmental Engineering, Stanford University, Stanford, CA 94305-4020, USA

^* To whom correspondence should be addressed. Email: qiaocl{at}ioz.ac.cn.

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the P=S group in the molecules was taxonomically identified as Klebsiella sp. strain F51-1-2. The gene involved in the reduction of organophosphorus compounds was cloned from this strain by shotgun technique and the deduced protein (named AKR5F1) showed homology to aldo-keto reductases (AKRs) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a, and overexpressed in E. coli BL21 (DE3). Recombinant His₆-tagged AKR5F1 was purified in one step using Ni-NTA affinity chromatography. Assays for cofactor specificity indicated that reductive transformation of organophosphorus compounds by the recombinant AKR5F1 specifically required NADH. The kinetic constants of the purified recombinant AKR5F1 were determined toward six thion organophosphorus compounds. For example, the K_m and k_cat values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5±47.0 µM and 25.7±1.7 min^-1, respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.