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

Jacob Ball,Francesca Salvi,Giovanni Gadda

doi:10.1074/jbc.m116.739151

Jacob Ball, Francesca Salvi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.739151

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Abstract

The protein PA1024 from Pseudomonas aeruginosa PAO1 is currently classified as 2-nitropropane dioxygenase, the previous name for nitronate monooxygenase in the GenBankTM 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 kred value of 24 s−1 and an apparent Kd 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 GenBankTM, the vast majority of which are currently misannotated as nitronate monooxygenase.

Highlights

The discrepancy between the rapid increase in the number of sequenced genomes of prokaryotes and the slower experimental determination of protein function has resulted in the presence of a large number of hypothetical proteins in the databases, with gene function prediction often unreliable [1, 2]
P. aeruginosa PAO1 possesses two other genes coding for hypothetical nitronate monooxygenases (NMOs), namely pa0660 and pa1024; these proteins do not carry the four motifs characteristic of class I NMO nor any of the motif signatures of class II NMO, raising the possibility that they code for enzymes with different function
Protein Purification—The gene pa1024 was cloned from the genomic DNA of P. aeruginosa PAO1 in the expression vector pET20b(ϩ), with the addition of a His tag at the C terminus of the recombinant protein

Summary

Introduction

The discrepancy between the rapid increase in the number of sequenced genomes of prokaryotes and the slower experimental determination of protein function has resulted in the presence of a large number of hypothetical proteins in the databases, with gene function prediction often unreliable [1, 2]. The recent structural and kinetic characterization of the gene product PA4202 from Pseudomonas aeruginosa PAO1 as Pa-NMO identified four motifs that establish class I NMO, with 500 sequences from bacteria, fungi, one insect, and one animal [6]. The structural characterization of PA1024 previously highlighted six motifs conserved in other hypothetical proteins similar to PA1024 [7], which are different from the four motifs described for class 1 NMO [6]. We hypothesized the reduced enzyme is oxidized by quinones because they are prevalent redox agents in nature [11, 12] To investigate this hypothesis, we experimentally tested NADH, NADPH, and various quinones for catalytic activity. The results reinforce the need for accurate experimental data on select hypothetical proteins to work in concert with computational methods for improved gene function prediction

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