title: Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides.
abstract: The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with K(m) and k(cat) values of 29.5 μM and 49.2 s(-1), respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing, Jiangsu, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Jin-Gang
authors.initials: JG
authors.lastname: Yin
authors.forename: Bao-Jian
authors.initials: BJ
authors.lastname: Hang
authors.forename: Shu
authors.initials: S
authors.lastname: Cai
authors.forename: Jian
authors.initials: J
authors.lastname: He
authors.forename: Shun-Gui
authors.initials: SG
authors.lastname: Zhou
authors.forename: Shun-Peng
authors.initials: SP
authors.lastname: Li
mesh_terms: D000577:Amides / Q000737:chemistry / Q000378:metabolism*; D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004132:Diflubenzuron / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010230:Paracoccus / Q000145:classification / Q000201:enzymology* / Q000235:genetics*; D010575:Pesticides / Q000737:chemistry / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D000577:Amides; D001426:Bacterial Proteins; D010575:Pesticides; D011994:Recombinant Proteins; D000581:Amidohydrolases; D004132:Diflubenzuron