Phylogenomics Reveal the Dynamic Evolution of Fungal Nitric Oxide Reductases and Their Relationship to Secondary Metabolism

Abstract

Fungi expressing P450nor, an unconventional nitric oxide (NO) reducing cytochrome P450, are considered significant contributors to environmental nitrous oxide (N2O) emissions. Despite extensive efforts, fungal contributions to N2O emissions remain uncertain. For example, the majority of N2O emitted from antibiotic-amended soil microcosms is attributed to fungal activity, yet axenic fungal cultures do not couple N-oxyanion respiration to growth and these fungi produce only minor quantities of N2O. To assist in reconciling these conflicting observations and produce a benchmark genomic analysis of fungal denitrifiers, genes underlying denitrification were examined in >700 fungal genomes. Of 167 p450nor—containing genomes identified, 0, 30, and 48 also harbored the denitrification genes narG, napA, or nirK, respectively. Compared with napA and nirK, p450nor was twice as abundant and exhibited 2–5-fold more gene duplications, losses, and transfers, indicating a disconnect between p450nor presence and denitrification potential. Furthermore, cooccurrence of p450nor with genes encoding NO-detoxifying flavohemoglobins (Spearman r = 0.87, p = 1.6e−10) confounds hypotheses regarding P450nor’s primary role in NO detoxification. Instead, ancestral state reconstruction united P450nor with actinobacterial cytochrome P450s (CYP105) involved in secondary metabolism (SM) and 19 (11%) p450nor-containing genomic regions were predicted to be SM clusters. Another 40 (24%) genomes harbored genes nearby p450nor predicted to encode hallmark SM functions, providing additional contextual evidence linking p450nor to SM. These findings underscore the potential physiological implications of widespread p450nor gene transfer, support the undiscovered affiliation of p450nor with fungal SM, and challenge the hypothesis of p450nor’s primary role in denitrification.

Nitric oxide as a developmental and metabolic signal in filamentous fungi

“…Recently, a phylogenetic study has revealed that the genes encoding P450 nor are not mainly involved in NO denitrification, but positioned in secondary metabolism gene clusters as synthetic genes in species from Tremellomycetes (i.e., Trichosporon asahii vs. ar asahii CBS2479 ), Leotiomycetes (i.e., Pseudogymnoascus destructans 20631‐21, Sclerotina sclerotiorum 1980 UF‐70 ), Dothideomycetes (i.e., Biopolaris sorokiniana ND90 Pr ) and Sordariomycetes (i.e., N. crassa OR74A, N. tetrasperma FGSC 2506, P. anserina S mat + ) (Figure ). Phylogenetic reconstruction of C‐type and a ketosynthase domain encoded by NRPS and PKS genes adjoining P450 nor has enabled the prediction of HC‐toxin ( 23 ) in species like Valetoniellopsis laxa and A. sojae ; Fumonisin ( 24 ) in Fusarium virguliform e and Magnaporthiopsis poa e; compactin ( 25 ), lovastatin ( 26 ), and epothilone A ( 27 ) in Penicillium citrinum and A. terreus ; and naphtopyrone ( 28 ), bikaverin ( 29 ) and aflatoxin B ( 30 ) in A. nidulans, A. parasiticus and Trichophyton soudanense by P450 nor ‐containing biosynthetic gene clusters (Higgins, Schadt, Matheny & Löffler, ). These data indicate that NO participates in fungal secondary metabolism not only as a signaling molecule, but also as one of the moieties to be integrated into the structure of metabolites or as one of the intermediates during the synthesis of fungal SM (Figures and ).…”

Algal photosynthesis converts nitric oxide into nitrous oxide

“…Based on ribonucleic acid interference (RNAi) silencing experiments in C. reinhardtii, Plouviez et al (13) concluded that the CYP55 homolog is the main contributor to N 2 O production. However, this gene has so far only been identified in three sequenced algal genomes (21), suggesting the occurrence of other mechanisms. C. reinhardtii FLVs have been recently shown to be involved in O 2 photoreduction (22), but have not been considered as catalyzing NO reduction so far (23).…”

NO and N2O transformations of diverse fungi in hypoxia: evidence for anaerobic respiration only in Fusarium strains

“…S2). Although the actual abiotic NO production rate of media under anoxia has to be tested for each medium to draw solid conclusions, our results suggest that fungal cultures with N 2 O rates below 2 nM ml −1 medium day −1 in amended media might actually not respire N‐oxyanions but instead use denitrification reductases or other enzymes to detoxify abiotically produced NO (Higgins et al ., ).…”

“…Current knowledge on fungal denitrification is mainly based on studies of Fusarium oxysporum (Shoun and Tanimoto, ; Zhou et al ., ; Shoun et al ., ). , and NO reductases were found ( napA , nirK and p450nor respectively) in fungi, but a nitrous oxide reductase has not been detected in any strain tested so far (Higgins et al ., ).…”