title: Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA.
abstract: The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.
authors.affiliation: Department of Microbiology, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea ykim@cu.ac.kr hanc210@snu.ac.kr.
authors.forename: Yong-Hak
authors.initials: YH
authors.lastname: Kim
authors.affiliation: Department of Microbiology, Catholic University of Daegu School of Medicine, Daegu, Republic of Korea.
authors.forename: Wooseok
authors.initials: W
authors.lastname: Song
authors.affiliation: Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea.
authors.forename: Jin-Sik
authors.initials: JS
authors.lastname: Kim
authors.affiliation: Department of Manufacturing Pharmacy, Pusan National University, Busan, Republic of Korea.
authors.forename: Li
authors.initials: L
authors.lastname: Jiao
authors.affiliation: Department of Life Science, Chung-Ang University, Seoul, Republic of Korea.
authors.forename: Kangseok
authors.initials: K
authors.lastname: Lee
authors.affiliation: Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, Center for Food and Bioconvergence, Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea ykim@cu.ac.kr hanc210@snu.ac.kr.
authors.forename: Nam-Chul
authors.initials: NC
authors.lastname: Ha
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D009579:Nitrobenzoates / Q000737:chemistry* / Q000378:metabolism*; D009601:Nitroreductases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D012965:Sodium Chloride / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: C518437:2-nitrobenzoate; D050505:Mutant Proteins; D009579:Nitrobenzoates; D012965:Sodium Chloride; D009601:Nitroreductases