title: Expression, characterization, and site-directed mutation of a multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) from Pseudomonas sp. Lm10.
abstract: A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonas putida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the K (m) value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.
authors.affiliation: College of Plant Protection, Nanjing Agricultural University, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Zhi-Fei
authors.initials: ZF
authors.lastname: Lang
authors.forename: Jing-Jing
authors.initials: JJ
authors.lastname: Shen
authors.forename: Shu
authors.initials: S
authors.lastname: Cai
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: He
authors.forename: Shun-Peng
authors.initials: SP
authors.lastname: Li
mesh_terms: D000095:Acetolactate Synthase / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism*; D015870:Gene Expression; D006540:Herbicides / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D006540:Herbicides; D050505:Mutant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000095:Acetolactate Synthase