C1-C2-nirS Domain Architecture is Unique to Chloroflexi
Though putative homologs exist independently for the constituent C1 and C2-NirS regions, respectively, these hits reflect different cytochrome or cytochrome-type nitrite reductases (largely in Proteobacteria, Nitrospirae, and Nitrospinae). The full C1-C2-NirS architecture appears unique to Chloroflexi, and is not observed in other groups. Querying NCBI’s non-redundant environmental database (env-nr) with the full ORF from SURF MAG 42 did not identify additional examples of the full gene construct. While several hits were identified that reflected putative homology to the joint C2-NirS domains, none of these included the C1 domain as well (Supplementary Datafile S2). An independent search of the env-nr database using the C1 domain as a query returned few overall hits. While some of these putative C1 homologs were identified in ORFs containing additional cytochrome-type enzyme superfamily domains or subunits, none co-occurred with NirS or NirK domains. These data suggest that there is little to no missing diversity of the Chloroflexi-type chimeric nitrite reductase in existing metagenomes.
Attempts to visualize the full enzyme structure using homology modeling (Bienert et al. , 2017; Waterhouse et al. , 2018) were unsuccessful; structural models were only able to predict a close match for the conserved C2-NirS region of the putative gene (Supplementary Datafile S3). Efforts to independently model the C1 structure could not recover predicted QMEAN scores above -4.50 (Benkert et al 2011). The poor scores may reflect the relatively short length of the cytochrome coding region. However, the Chloroflexi nirS gene sequence does retain several conserved residues present in the crystal structure of Pseudomonas aeruginosa NirS. In P. aeruginosaNirS, His51 and Met88 coordinate heme c; His182 coordinates heme d1; and His327 and His369 are believed to stabilize the active site nitrite anion (Rinaldo et al. , 2011; Maia and Moura, 2014). Corresponding residues are conserved within the C2 (His65, Met125) and NirS alignments (His46, His239, His300) for the Chloroflexi NirS ORF; interestingly, the residue corresponding to His327 (His239) is not universally conserved, though it is conserved among Chloroflexi with the novel NirS architecture (Supplementary Datafile S1).