Kim, Yong-Hak Song, Woo-Seok Go, Hayoung Cha, Chang-Jun Lee, Cheolju Yu, Myeong-Hee Lau, Peter C. K. Lee, Kangseok 2024-01-20T13:04:50Z 2024-01-20T13:04:50Z 2021-09-01 2013-01 0021-9193 https://pubs.kist.re.kr/handle/201004/128514 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 Delta 65-74 exhibited markedly reduced activity toward 2,4-DNBA, but its 2-NBA reduction activity was unaffected; however, both activities were abolished in the Delta 193-216 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 ZnCl2, 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 H2O2. 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. English AMER SOC MICROBIOLOGY INDUCED CONFORMATIONAL-CHANGES ESCHERICHIA-COLI ARTHROBACTER-PROTOPHORMIAE GLUTAMINE-SYNTHETASE DEGRADATION PATHWAY O-NITROBENZOATE PROTEIN 3-HYDROXYANTHRANILATE BIODEGRADATION IDENTIFICATION 2-Nitrobenzoate 2-Nitroreductase (NbaA) Switches Its Substrate Specificity from 2-Nitrobenzoic Acid to 2,4-Dinitrobenzoic Acid under Oxidizing Conditions Article 10.1128/JB.02016-12 1 JOURNAL OF BACTERIOLOGY, v.195, no.2, pp.180 - 192 JOURNAL OF BACTERIOLOGY 195 2 180 192 scie scopus 000316959600002 2-s2.0-84872122308 Microbiology Microbiology Article INDUCED CONFORMATIONAL-CHANGES ESCHERICHIA-COLI ARTHROBACTER-PROTOPHORMIAE GLUTAMINE-SYNTHETASE DEGRADATION PATHWAY O-NITROBENZOATE PROTEIN 3-HYDROXYANTHRANILATE BIODEGRADATION IDENTIFICATION