Nitrate reduction byDesulfovibrio desulfuricans: A periplasmic nitrate reductase system that lacks NapB, but includes a unique tetrahemec-type cytochrome, NapM

Abstract

Many sulphate reducing bacteria can also reduce nitrite, but relatively few isolates are known to reduce nitrate. Although nitrate reductase genes are absent from Desulfovibrio vulgaris strain Hildenborough, for which the complete genome sequence has been reported, a single subunit periplasmic nitrate reductase, NapA, was purified from Desulfovibrio desulfuricans strain 27774, and the structural gene was cloned and sequenced. Chromosome walking methods have now been used to determine the complete sequence of the nap gene cluster from this organism. The data confirm the absence of a napB homologue, but reveal a novel six-gene organisation, napC-napM-napA-napD-napG-napH. The NapC polypeptide is more similar to the NrfH subgroup of tetraheme cytochromes than to NapC from other bacteria. NapM is predicted to be a tetra-heme c-type cytochrome with similarity to the small tetraheme cytochromes from Shewanella oneidensis. The operon is located close to a gene encoding a lysyl-tRNA synthetase that is also found in D. vulgaris. We suggest that electrons might be transferred to NapA either from menaquinol via NapC, or from other electron donors such as formate or hydrogen via the small tetraheme cytochrome, NapM. We also suggest that, despite the absence of a twin-arginine targeting sequence, NapG might be located in the periplasm where it would provide an alternative direct electron donor to NapA.

The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB

“…The function of NapC in NAP in these species and in S. oneidensis MR-1, which also lacks NapC, may be met by the periplasmic c-type cytochrome homologue CymA, which is also from the NapC/NirT family (Gao et al, 2009;Murphy & Saltikov, 2007;Myers & Myers, 1997, 2000Schwalb et al, 2003). The napCMADGH operon of the d-proteobacterium Desulfovibrio desulfuricans lacks the napB gene (Marietou et al, 2005). Without the napB gene, NAP from D. desulfuricans is expressed as a catalytic monomer, NapA (Dias et al, 1999;Najmudin et al, 2008).…”

“…At least 11 different types of nap gene clusters have been described from bacteria (González et al, 2006;Marietou et al, 2005;Richardson et al, 2001). The napA gene encodes the catalytic NapA subunit where nitrate reduction takes place and which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor (Mo-bis-MGD) and one [4Fe24S] iron-sulfur cluster.…”

“…The napDABC genes are found in a-, b-and c-proteobacteria (Berks et al, 1995;Delgado et al, 2003;Hettmann et al, 2004;Reyes et al, 1998) and encode what were once defined as the 'essential' NAP proteins (Potter & Cole, 1999), with NapC being a membrane-anchored tetra-haem c-type cytochrome of the NapC/NirT family that mediates electron transport from the quinol pool to NapB (Cartron et al, 2002;Roldan et al, 1998) and NapD being a maturation chaperone for NapA. The napDABC gene cluster is interrupted by genes encoding an iron-sulfurcluster-based NapGH complex in the napFDAGHBC operon of the c-proteobacteria Escherichia coli (Grove et al, 1996), Haemophilus influenzae and Salmonella typhimurium (Marietou et al, 2005) and the a-proteobacterium Magnetospirillum magnetotacticum (Taoka et al, 2003). The nap operons of the e-proteobacteria Wolinella succinogenes (napAGHBFLD) (Kern & Simon, 2008;Simon et al, 2003) and Campylobacter jejuni (napAGHBLD) do not include the napC gene.…”

“…Soluble NAP or membrane-bound nitrate reductase (NAR) systems manage catabolic respiratory nitrate reduction and the assimilatory nitrate reductase (NAS) system governs ammonium production in the cytoplasm for anabolic purposes (González et al, 2006;Richardson et al, 2001;Stolz & Basu, 2002;Tavares et al, 2006;Zumft, 1997). As currently understood, the two respiratory pathways are distinguished downstream of the reduction of nitrate to nitrite by the enzymes that catalyse either the six-electron reduction of nitrite to ammonium (cytochrome c nitrite reductase, NrfA) in respiratory ammonification, or the step-wise reduction of nitrite to nitric oxide (copper nitrite reductase, NirK; or cytochrome cd 1 nitrite reductase, NirS), nitric oxide to nitrous oxide (nitric oxide reductase, NOR) and nitrous oxide to dinitrogen (nitrous oxide reductase, NOS) in denitrification (González et al, 2006;Richardson et al, 2001;Stolz & Basu, 2002;Tavares et al, 2006;Zumft, 1997).At least 11 different types of nap gene clusters have been described from bacteria (González et al, 2006;Marietou et al, 2005;Richardson et al, 2001). The napA gene encodes the catalytic NapA subunit where nitrate reduction takes place and which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor (Mo-bis-MGD) and one [4Fe24S] iron-sulfur cluster.…”

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

“…A 3.2-Å resolution structure of this enzyme suggests that tight complex formation is contributed to by two loops at the N-and C-terminal extremities of NapB that adopt an extended conformation and embrace the NapA subunit (10). However, another structure of NapA, the 1.9 Å resolution structure from the ␦-proteobacterium Desulfovibrio desulfuricans, is that of a monomer, and there is currently no evidence that this enzyme ever forms a heterodimer with a NapB subunit (9,16,17). This has led to a subclassification of periplasmic nitrate reductases into monomeric and heterodimeric members (10).…”