4Z85

Crystal structur of Pseudomonas fluorescens 2-nitrobenzoate 2-nitroreductase NbaA

Summary for 4Z85

• Entry DOI: 10.2210/pdb4z85/pdb
• Descriptor: 2-nitrobenzoate nitroreductase (2 entities in total)
• Functional Keywords: 2-nitrobenzoate 2-nitroreductase (nbaa), 2-nitrobenzoate methyl ester, fmn-binding site, tyrosine modification, cysteine reactivity, oxidoreductase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 24437.93

Authors

Ha, N.C.,Jiao, L.,Kim, J.S. (deposition date: 2015-04-08, release date: 2016-01-20, Last modification date: 2023-11-08)

Primary citation

Kim, Y.H.,Song, W.,Kim, J.S.,Jiao, L.,Lee, K.,Ha, N.C.
Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA
Appl.Environ.Microbiol., 81:5266-5277, 2015

PubMed 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.

PubMed: 26025888
DOI: 10.1128/AEM.01289-15

Experimental method

X-RAY DIFFRACTION (1.7 Å)