Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112.
The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.
Wu
Pei C
PC
State Key Laboratory of Biocontrol, School of Life Science, Zhongshan University, Guangzhou 510275, People's Republic of China.
Liu
Yu H
YH
Wang
Zhuo Y
ZY
Zhang
Xiao Y
XY
Li
He
H
Liang
Wei Q
WQ
Luo
Na
N
Hu
Ji M
JM
Lu
Jia Q
JQ
Luan
Tian G
TG
Cao
Li X
LX
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007709:Klebsiella / Q000201:enzymology*; D008969:Molecular Sequence Data; D011722:Pyrethrins / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
D011722:Pyrethrins; D004950:Esterases