2-Nitrobenzoate 2-Nitroreductase (NbaA) switches its substrate specificity from 2-Nitrobenzoic acid to 2,4-Dinitrobenzoic acid under oxidizing conditions

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DOIResolve DOI: http://doi.org/10.1128/JB.02016-12
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TypeArticle
Journal titleJournal of Bacteriology
ISSN0021-9193
Volume195
Issue2
Pages480192; # of pages: 289
Subject2 nitrobenzoate 2 nitroreductase; 2 nitrobenzoic acid; 2,4 dinitrobenzoic acid; cysteine; dithiothreitol; nitrobenzoic acid derivative; nitroreductase; oligomer; reduced nicotinamide adenine dinucleotide phosphate; unclassified drug; anaerobic capacity; article; catalysis; disulfide bond; enzyme activity; enzyme modification; high performance liquid chromatography; nonhuman; nucleotide sequence; pH; polyacrylamide gel electrophoresis; priority journal; tandem mass spectrometry; temperature; thin layer chromatography; Pseudomonas fluorescens
Abstract2-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 reductionactivity 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 1mMdithiothreitol 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 higherorder 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. © 2013, American Society for Microbiology.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); NRC Biotechnology Research Institute (BRI-IRB)
Peer reviewedYes
NPARC number21269595
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Record identifierb0fd7e88-20f3-490a-81c1-b1203bdfa965
Record created2013-12-13
Record modified2016-05-09
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