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Applied and Environmental Microbiology, August 2007, p. 4959-4965, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.02993-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Hong Jiang,1,{dagger} Chao Yang,1,2,{dagger} Hong Qu,3 Zheng Liu,1,2 Q. S. Fu,4 and Chuanling Qiao1*

State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China,1 Graduate School of the Chinese Academy of Sciences, Beijing 100049, China,2 College of Life Sciences, Peking University, Beijing 100871, China,3 Department of Civil and Environmental Engineering, Stanford University, Stanford, California 94305-40204

Received 26 December 2006/ Accepted 31 May 2007

A soil bacterium capable of metabolizing organophosphorus compounds by reducing the PFormula 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 the shotgun technique, and the deduced protein (named AKR5F1) showed homology to members of the aldo-keto reductase (AKR) superfamily. The intact coding region for AKR5F1 was subcloned into vector pET28a and overexpressed in Escherichia coli BL21(DE3). Recombinant His6-tagged AKR5F1 was purified in one step using Ni-nitrilotriacetic acid 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 toward six thion organophosphorus compounds were determined. For example, the Km and kcat 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.

* Corresponding author. Mailing address: State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. Phone: 86-10-64807191. Fax: 86-10-64807099. E-mail: qiaocl{at}ioz.ac.cn

{triangledown} Published ahead of print on 15 June 2007.

{dagger} H.J. and C.Y. contributed equally to this work.

Applied and Environmental Microbiology, August 2007, p. 4959-4965, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.02993-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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