Cloning of a novel aldo-keto reductase gene from Klebsiella sp. strain F51-1-2 and its functional expression in Escherichia coli.
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 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 His(6)-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 K(m) and k(cat) values of reductive transformation of malathion by the purified recombinant AKR5F1 are 269.5 +/- 47.0 microM and 25.7 +/- 1.7 min(-1), respectively. Furthermore, the reductive transformation of organophosphorus compounds can be largely explained by structural modeling.
Jiang
Hong
H
State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Yang
Chao
C
Qu
Hong
H
Liu
Zheng
Z
Fu
Q S
QS
Qiao
Chuanling
C
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002800:Cholinesterase Inhibitors / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007709:Klebsiella / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D008294:Malathion / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds / Q000378:metabolism; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002800:Cholinesterase Inhibitors; D009943:Organophosphorus Compounds; D012336:RNA, Ribosomal, 16S; D000429:Alcohol Oxidoreductases; D000449:Aldehyde Reductase; D008294:Malathion