Abstract
Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.
Highlights
Nitrate is one of the major inorganic nitrogen sources for microbes
We report the characterization of a novel nitrate reductase (NAS) encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution
Previous genetic studies showed that neither the narGHJInor the narB-encoded putative nitrate reductase (NaR) enzyme was responsible for M. smegmatis nitrate assimilation and suggested that the proposed NAS ought to be another molybdopterin cofactor (MoCo)-containing enzyme [18, 19]
Summary
NaR, nitrate reductase; NAS, assimilatory nitrate reductase; CA domain, the nitrate reduction catalytic domain; DF domain, the FMN–FAD/NAD domain; Fv/Fd-NarB, flavodoxin/ferredoxin-dependent heterodimeric NarB; MoCo, molybdopterin cofactor; N-NasN, N-termi-. The holoenzymes of all prokaryotic NASs characterized to date are highly evolutionarily conserved with respect to their catalytic subunit, which contains a canonical nitrate reduction catalytic domain (CA domain), consisting of three binding subdomains bound with a [4Fe-4S] cluster, a molybdopterin, and a bis-molybdopterin guanine dinucleotide, a form of molybdopterin cofactor (MoCo) (Fig. 1) These NAS enzymes are significantly different with respect to the intermolecular transfer of electrons derived from their respective electron donors to the catalytic sites. We have identified that M. smegmatis NAS is encoded by the Msmeg_4206 gene and is essential for M. smegmatis to assimilate nitrate under aerobic conditions It is characterized as an NADPH-dependent monomeric NAS containing a canonical CA domain and an FMN–FAD/ NAD electron-receiving/transferring domain (DF domain) identified in the diflavin reductase family, and it is designated NasN. Unlike their fastgrowing environmental ancestors, most of the slow-growing pathogenic mycobacteria have lost nasN via a few independent genomic deletion events
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