NqrM (DUF539) Protein Is Required for Maturation of Bacterial Na ⁺ -Translocating NADH:Quinone Oxidoreductase

Abstract

Na؉ -translocating NADH:quinone oxidoreductase (Na ؉ -NQR) catalyzes electron transfer from NADH to ubiquinone in the bacterial respiratory chain, coupled with Na ؉ translocation across the membrane. Na ؉ -NQR maturation involves covalent attachment of flavin mononucleotide (FMN) residues, catalyzed by flavin transferase encoded by the nqr-associated apbE gene. Analysis of complete bacterial genomes has revealed another putative gene (duf539, here renamed nqrM) that usually follows the apbE gene and is present only in Na ؉ -NQR-containing bacteria. Expression of the Vibrio harveyi nqr operon alone or with the associated apbE gene in Escherichia coli, which lacks its own Na ؉ -NQR, resulted in an enzyme incapable of Na ؉ -dependent NADH or reduced nicotinamide hypoxanthine dinucleotide (dNADH) oxidation. However, fully functional Na ؉ -NQR was restored when these genes were coexpressed with the V. harveyi nqrM gene. Furthermore, nqrM lesions in Klebsiella pneumoniae and V. harveyi prevented production of functional Na ؉ -NQR, which could be recovered by an nqrM-containing plasmid. The Na ؉ -NQR complex isolated from the nqrM-deficient strain of V. harveyi lacks several subunits, indicating that nqrM is necessary for Na ؉ -NQR assembly. The protein product of the nqrM gene, NqrM, contains a single putative transmembrane ␣-helix and four conserved Cys residues. Mutating one of these residues (Cys33 in V. harveyi NqrM) to Ser completely prevented Na ؉ -NQR maturation, whereas mutating any other Cys residue only decreased the yield of the mature protein. These findings identify NqrM as the second specific maturation factor of Na ؉ -NQR in proteobacteria, which is presumably involved in the delivery of Fe to form the (Cys) 4 [Fe] center between subunits NqrD and NqrE. IMPORTANCE Na؉ -translocating NADH:quinone oxidoreductase complex (Na ؉ -NQR) is a unique primary Na ؉ pump believed to enhance the vitality of many bacteria, including important pathogens such as Vibrio cholerae, Vibrio parahaemolyticus, Haemophilus influenzae, Neisseria gonorrhoeae, Pasteurella multocida, Porphyromonas gingivalis, Enterobacter aerogenes, and Yersinia pestis. Production of Na؉ -NQR in bacteria requires Na ؉ -NQR-specific maturation factors. We earlier identified one such factor (ApbE) that covalently attaches flavin residues to Na ؉ -NQR. Here we identify the other protein factor, designated NqrM, and show that NqrM and ApbE suffice to produce functional Na ؉ -NQR from the Vibrio harveyi nqr operon. NqrM may be involved in Fe delivery to a unique Cys 4 [Fe] center during Na ؉ -NQR assembly. Besides highlighting Na ؉ -NQR biogenesis, these findings suggest a novel drug target to combat Na ؉ -NQR-containing bacteria.N a ϩ -translocating NADH:quinone oxidoreductase (Na ϩ -NQR) is a redox-driven sodium pump operating in the respiratory chains of various bacteria, including several pathogens (1). This enzyme is a functional analog of H ϩ -translocating NADH: quinone oxidoreductase, known as mitochondrial complex I or bacterial NDH-1...

Sodium antiporters of Pseudomonas aeruginosa in challenging conditions: effects on growth, biofilm formation, and swarming motility

“…The primary transporters NQR and Complex I were studied, as well as the secondary antiporters Mrp, NhaB, and NhaP. The loss of the NQR did not affect growth under challenging sodium and pH conditions, which confirms a recent study by Raba et al [37] who suggested that in contrast to NQR in other species, such as Vibrio [3,23,27,51], Pa-NQR is a proton pump with no affinity for sodium. In addition to that, our results suggest that loss of NQR does not affect pH homeostasis, biofilm formation, or swarming motility, which might be due to complementary proton pumping activity of Complex I.…”

Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

“…It is necessary for NQR maturation, since the FMNs are required for the transfer of the electrons through the NQR complex. NqrM was predicted to deliver Fe to subunits NqrD and NqrE ( Kostyrko et al, 2015 ). Subunits NqrD and NqrE harbor an iron cluster located in the midst of the membrane part of NQR which is proposed to participate in intramolecular electron transfer ( Steuber et al, 2014b ).…”

Development of a novel rationally designed antibiotic to inhibit a nontraditional bacterial target

“…Conversely, the data with PEG-2 and PEG-2S support this agent as an effective inhibitor of Na + -NQR. The experiments with subbacterial membrane vesicles allowed the measurement of Na + -NQR activity directly when the enzyme was operating in a physiologically relevant background (being placed in its native membrane and supplying electrons taken from NADH to the respiratory chain) but without additional permeability barriers and influences from cytoplasmic metabolism (Bertsova and Bogachev 2004;Fadeeva et al 2007Fadeeva et al , 2008; Kostyrko et al 2016). In this model, PEG-2S exhibited a low MIC 50 of 1.76 nmol/L (Fig.…”

“…Na + -NQR activity can be differentially measured in sub-bacterial membrane vesicles as the NQR-mediated oxidation of an NADH analog, dNADH (Bertsova and Bogachev 2004;Fadeeva et al 2007Fadeeva et al , 2008; Kostyrko et al 2016). This allows the measurement of NQR activity directly without confounding factors -the enzyme is functioning in its native membrane and feeding the electrons taken from NADH to the respiratory chain.…”