F420H2-Dependent Degradation of Aflatoxin and other Furanocoumarins Is Widespread throughout the Actinomycetales

Abstract

Two classes of F 420 -dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis . One class, the FDR-A enzymes, has up to 100 times more activity than the other. F 420 is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria . We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales , finding that nine can also use F 420 H 2 to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales . The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F 420 to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…Bacteria likewise appear to tightly couple substrate oxidation (glucose-6-phosphate and NADPH) to F 420 reduction, presumably to en- hance catalytic efficiency (Table 1). Partly due to its low redox potential, the F 420 H 2 produced is capable of reducing a wide range of organic compounds otherwise recalcitrant to activation as discussed in section 4 (28,54,55). Recent work also indicates that F 420 may be utilized in aerobic bacteria in hypoxic and anoxic environments, potentially substituting for high-potential nicotinamide cofactors (NAD and NADP) (Ϫ320 mV) (30,32,56).…”

“…There is also evidence that mycobacteria instead use electrons liberated from G6P by Fgd to directly detoxify exogenous agents (363). Two independent studies have demonstrated that FDOR-As rapidly reduce menadione and plumbagin using F 420 H 2 (30,32), and it is also plausible that these highly promiscuous proteins (28,55) can directly detoxify certain antibiotics too. However, genetic studies have yet to definitively link FDORs to antibiotic resistance and oxidative stress responses.…”

“…While polynitroaromatic compounds are anthropogenic, actinobacteria may have evolved the capacity to degrade them from preexisting pathways that metabolize naturally occurring nitroaromatic compounds (e.g., chloramphenicol) (399,400). It has also been demonstrated that F 420 H 2 -dependent oxidoreductases of the flavin/ deazaflavin oxidoreductase superfamily have broad substrate specificity; enzymes purified from genera as diverse as Mycobacterium, Frankia, Nocardia, and Rhodococcus are capable of reducing coumarin natural products (28,55). F 420 may also contribute to the well-reported abilities of soil actinomycetes to biodegrade a wide variety of other polycyclic aromatic hydrocarbons (401).…”

“…18). Proteins from the FDOR-A family are exclusively F 420 -binding proteins (28,35,55,164,410) restricted to the phyla Actinobacteria and Chloroflexi (28,30,37). In contrast, FDOR-B proteins are widely distributed, including in bacteria that do not synthesize F 420 .…”

The methanogenic redox cofactor F420 is widely synthesized by aerobic soil bacteria

“…Among them are the one-carbon reactions of methanogenesis (Thauer, 1998;Shima et al, 2000;Hagemeier et al, 2003), the biosynthesis pathways of tetracycline antibiotics (Wang et al, 2013) and the biodegradation of picrate and aflatoxins (Ebert et al, 2001;Taylor et al, 2010;Lapalikar et al, 2012). The cofactor appears to have been selected for these roles because of its unique electrochemical properties compared with the ubiquitous flavin and nicotinamide cofactors FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide) and NAD(P) (nicotinamide adenine dinucleotide (phosphate)) (Walsh, 1986;Greening et al, 2016a).…”