title: 2-nitrobenzoate 2-nitroreductase (NbaA) switches its substrate specificity from 2-nitrobenzoic acid to 2,4-dinitrobenzoic acid under oxidizing conditions.
abstract: 2-Nitrobenzoate 2-nitroreductase (NbaA) of Pseudomonas fluorescens strain KU-7 is a unique enzyme, transforming 2-nitrobenzoic acid (2-NBA) and 2,4-dinitrobenzoic acid (2,4-DNBA) to the 2-hydroxylamine compounds. Sequence comparison reveals that NbaA contains a conserved cysteine residue at position 141 and two variable regions at amino acids 65 to 74 and 193 to 216. The truncated mutant Δ65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Δ193-216 mutant, suggesting that these regions are necessary for the catalysis and specificity of NbaA. NbaA showed different lag times for the reduction of 2-NBA and 2,4-DNBA with NADPH, and the reduction of 2,4-DNBA, but not 2-NBA, failed in the presence of 1 mM dithiothreitol or under anaerobic conditions, indicating oxidative modification of the enzyme for 2,4-DNBA. The enzyme was irreversibly inhibited by 5,5'-dithio-bis-(2-nitrobenzoic acid) and ZnCl(2), which bind to reactive thiol/thiolate groups, and was eventually inactivated during the formation of higher-order oligomers at high pH, high temperature, or in the presence of H(2)O(2). SDS-PAGE and mass spectrometry revealed the formation of intermolecular disulfide bonds by involvement of the two cysteines at positions 141 and 194. Site-directed mutagenesis indicated that the cysteines at positions 39, 103, 141, and 194 played a role in changing the enzyme activity and specificity toward 2-NBA and 2,4-DNBA. This study suggests that oxidative modifications of NbaA are responsible for the differential specificity for the two substrates and further enzyme inactivation through the formation of disulfide bonds under oxidizing conditions.
authors.affiliation: Department of Microbiology, Catholic University of Daegu School of Medicine, Nam-Gu, Daegu, Republic of Korea. ykim@cu.ac.kr
authors.forename: Yong-Hak
authors.initials: YH
authors.lastname: Kim
authors.forename: Woo-Seok
authors.initials: WS
authors.lastname: Song
authors.forename: Hayoung
authors.initials: H
authors.lastname: Go
authors.forename: Chang-Jun
authors.initials: CJ
authors.lastname: Cha
authors.forename: Cheolju
authors.initials: C
authors.lastname: Lee
authors.forename: Myeong-Hee
authors.initials: MH
authors.lastname: Yu
authors.forename: Peter C K
authors.initials: PC
authors.lastname: Lau
authors.forename: Kangseok
authors.initials: K
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009579:Nitrobenzoates / Q000378:metabolism*; D009601:Nitroreductases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: C518437:2-nitrobenzoate; D004220:Disulfides; D050505:Mutant Proteins; D009579:Nitrobenzoates; D009249:NADP; C109832:2,4-dinitrobenzoic acid; D006861:Hydrogen Peroxide; D009601:Nitroreductases