title: Identification of the key amino acid sites of the carbendazim hydrolase (MheI) from a novel carbendazim-degrading strain Mycobacterium sp. SD-4.
abstract: A novel carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) degrading strain SD-4 was isolated and identified preliminarily as Mycobacterium sp. according to its phenotypic features and phylogenetic analysis. This strain could utilize MBC as the sole carbon and nitrogen sources for growth and degrade 50mgL-1MBC at the average degradation rate of 0.63mgL-1h-1. Strain SD-4 degraded MBC through the typical pathway, in which MBC was first hydrolyzed by MheI to 2-aminobenzimidazole (2-AB) and then converted to 2-hydroxybenzimidazole (2-HB). The MBC hydrolase encoding gene mheI was cloned from strain SD-4 and successfully expressed in Escherichia coli by codon optimization. The sulfhydryl-blocking assay revealed that the activity of MheI was closely related to cysteine, and the site-directed mutation experiment showed that Cys16 and Cys222 played important roles during the hydrolysis of MBC by MheI. Therefore they affected its activity directly and were defined as the key amino acid sites.
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Yingkun
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Wang
authors.affiliation: Industrial Product Division, Intrexon Corporation, South San Francisco, CA, 94080, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Hu
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Chenfei
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Wen
authors.initials: W
authors.lastname: Jin
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China.
authors.forename: Shijun
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China; Laboratory Centre of Life Science, College of Life Sciences, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China.
authors.forename: Gang
authors.initials: G
authors.lastname: Hu
authors.affiliation: Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture, College of life sciences, Nanjing Agricultural University, Nanjing, 210095, PR China. Electronic address: hongqing@njau.edu.cn.
authors.forename: Qing
authors.initials: Q
authors.lastname: Hong
mesh_terms: D001562:Benzimidazoles / Q000378:metabolism*; D002219:Carbamates / Q000378:metabolism*; D004926:Escherichia coli; D005798:Genes, Bacterial; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009161:Mycobacterium / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001562:Benzimidazoles; D002219:Carbamates; D006867:Hydrolases; C006698:carbendazim
keywords: Carbendazim; Expression; Key amino acid sites; Mycobacterium sp. SD-4; mheI