Functional Annotation of a Presumed Nitronate Monoxygenase Reveals a New Class of NADH:Quinone Reductases

Abstract

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBank TM and PDB databases, but the enzyme was not kinetically characterized. In this study, PA1024 was purified to high levels, and the enzymatic activity was investigated by spectroscopic and polarographic techniques. Purified PA1024 did not exhibit nitronate monooxygenase activity; however, it displayed NADH:quinone reductase and a small NADH:oxidase activity. The enzyme preferred NADH to NADPH as a reducing substrate. PA1024 could reduce a broad spectrum of quinone substrates via a Ping Pong Bi Bi steady-state kinetic mechanism, generating the corresponding hydroquinones. The reductive half-reaction with NADH showed a k red value of 24 s ؊1 and an apparent K d value estimated in the low micromolar range. The enzyme was not able to reduce the azo dye methyl red, routinely used in the kinetic characterization of azoreductases. Finally, we revisited and modified the existing six conserved motifs of PA1024, which define a new class of NADH:quinone reductases and are present in more than 490 hypothetical proteins in the GenBank TM , the vast majority of which are currently misannotated as nitronate monooxygenase.

Microbial communities and gene contributions in smokeless tobacco products

“…We underscore that this gene may represent an additional mechanism to generate nitrite and consequently TSNAs. However, we also note that annotations for NMO have been identified as inaccurate for some sequences in the database and in need of revision before actual metabolic activity can be definitively assigned (Ball et al 2016).…”

Proposal of the reverse flow model for the origin of the eukaryotic cell based on comparative analyses of Asgard archaeal metabolism

“…However, it is possible that the acquisition of mitochondria was an intermediate event that occurred after the invention of a basic intermembrane-system and actin cytoskeleton but predated the origin of the nucleus 54 . Furthermore, it remains an open question whether membrane lipids could have been of mixed origin during the transition phase 55 and were subsequently replaced by bacterial fatty-acid-type lipids in the outer membrane, following endosymbiont gene transfer 56 , gene acquisitions from other sources 57,58 and differential gene loss. Bacterial lipids may have had a selective advantage considering that genes for most metabolic enzymes, including respiratory chains, were retained from the endosymbiont rather than the host and were likely functioning optimally in bacterial membrane lipids.…”

“…Thorarchaeota seem to encode a multisubunit NADH dehydrogenase (Complex 1) (Suppl. 55 . Interestingly, at least one PF03060 domain protein of each Loki-and Thorarchaeota is much more similar to the NADH:quinone reductases of Pseudomonas aeruginosa PAO1 (PA1024) (ca.…”

In silico analyses of maleidride biosynthetic gene clusters

“…Six motifs which define this class of enzyme are almost completely conserved within the maleidride sequences (Figure S45). 68 Inspection of the structure of glauconic acid 2 does not reveal an obvious activity for a quinone reductase within this pathway.…”

“…An alignment with the known NADH dependent quinone reductase PA1024 (Q9I4V0.1) 68 shows high homology, with 51.99 -52.91 % sequence identity (Table S14). Six motifs which define this class of enzyme are almost completely conserved within the maleidride sequences (Figure S45).…”