title: The combined structural and kinetic characterization of a bacterial nitronate monooxygenase from Pseudomonas aeruginosa PAO1 establishes NMO class I and II.
abstract: Nitronate monooxygenase (NMO) oxidizes the mitochondrial toxin propionate 3-nitronate (P3N) to malonate semialdehyde. The enzyme has been previously characterized biochemically in fungi, but no structural information is available. Based on amino acid similarity 4,985 genes are annotated in the GenBank(TM) as NMO. Of these, 4,424 (i.e. 89%) are bacterial genes, including several Pseudomonads that have been shown to use P3N as growth substrate. Here, we have cloned and expressed the gene pa4202 of Pseudomonas aeruginosa PAO1, purified the resulting protein, and characterized it. The enzyme is active on P3N and other alkyl nitronates, but cannot oxidize nitroalkanes. P3N is the best substrate at pH 7.5 and atmospheric oxygen with k(cat)(app)/K(m)(app) of 12 × 10(6) M(-1) s(-1), k(cat)(app) of 1300 s(-1), and K(m)(app) of 110 μm. Anerobic reduction of the enzyme with P3N yields a flavosemiquinone, which is formed within 7.5 ms, consistent with this species being a catalytic intermediate. Absorption spectroscopy, mass spectrometry, and x-ray crystallography demonstrate a tightly, non-covalently bound FMN in the active site of the enzyme. Thus, PA4202 is the first NMO identified and characterized in bacteria. The x-ray crystal structure of the enzyme was solved at 1.44 Å, showing a TIM barrel-fold. Four motifs in common with the biochemically characterized NMO from Cyberlindnera saturnus are identified in the structure of bacterial NMO, defining Class I NMO, which includes bacterial, fungal, and two animal NMOs. Notably, the only other NMO from Neurospora crassa for which biochemical evidence is available lacks the four motifs, defining Class II NMO.
authors.affiliation: From the Departments of Chemistry.
authors.forename: Francesca
authors.initials: F
authors.lastname: Salvi
authors.affiliation: Biology.
authors.forename: Johnson
authors.initials: J
authors.lastname: Agniswamy
authors.affiliation: From the Departments of Chemistry.
authors.forename: Hongling
authors.initials: H
authors.lastname: Yuan
authors.affiliation: the Department of Bioengineering Sciences, Vrije Universiteit Brussel, Belgium, and the Department of Structural Biology Brussels, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Ken
authors.initials: K
authors.lastname: Vercammen
authors.affiliation: the Department of Bioengineering Sciences, Vrije Universiteit Brussel, Belgium, and the Department of Structural Biology Brussels, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Rudy
authors.initials: R
authors.lastname: Pelicaen
authors.affiliation: the Department of Bioengineering Sciences, Vrije Universiteit Brussel, Belgium, and the Department of Structural Biology Brussels, VIB, Pleinlaan 2, 1050 Brussels, Belgium.
authors.forename: Pierre
authors.initials: P
authors.lastname: Cornelis
authors.affiliation: the School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30302.
authors.forename: Jim C
authors.initials: JC
authors.lastname: Spain
authors.affiliation: From the Departments of Chemistry, Biology, Center for Biotechnology and Drug Design, iweber@gsu.edu.
authors.forename: Irene T
authors.initials: IT
authors.lastname: Weber
authors.affiliation: From the Departments of Chemistry, Biology, Center for Biotechnology and Drug Design, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, Georgia 30302, ggadda@gsu.edu.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gadda
mesh_terms: D000595:Amino Acid Sequence; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D006899:Mixed Function Oxygenases
keywords: 3-Nitropropionate; Enzyme Catalysis; Enzyme Structure; Flavoprotein; Nitronate Monooxygenase; Oxidoreductase; Propionate 3-Nitronate; Pseudomonas Aeruginosa (P. Aeruginosa); TIM Barrel; Toxin