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.
Highlights
The annotation of Ͼ4,900 genes for nitronate monooxygenase, which detoxifies a deadly toxin, is inaccurate due to the lack of functional data
The consensus motifs are found in 475 sequences annotated as hypothetical Nitronate monooxygenase (NMO) belonging to bacteria, fungi, and two animals, establishing a new class of enzymes based on functional annotation of a reference standard protein for which structural-functional information is available
Purification of Recombinant Pa-NMO—Pa-NMO was expressed in the E. coli strain Rosetta(DE3)pLysS and purified to a high level as judged from SDS-PAGE analysis (Fig. 1)
Summary
The annotation of Ͼ4,900 genes for nitronate monooxygenase, which detoxifies a deadly toxin, is inaccurate due to the lack of functional data. Functional annotation of prokaryotic genes based on experimental evidence represents only 0.33% of the microbial genes in COMBREX (COmputational BRidges to EXperiments) [1, 2] This is primarily due to the rapid progress in the sequencing of entire genomes that supersedes by far the biochemical characterization of the gene products [1, 2]. This problem is exacerbated by the limited accuracy of the computational prediction of function based on amino acid sequences [1, 2], mainly due to the paucity of biochemical information available for the reference sequence.
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