The multidomain flavodiiron protein from Clostridium difficile 630 is an NADH:oxygen oxidoreductase

Abstract

Flavodiiron proteins (FDPs) are enzymes with a minimal core of two domains: a metallo-β-lactamase-like, harbouring a diiron center, and a flavodoxin, FMN containing, domains. FDPs are O2 or NO reducing enzymes; for many pathogens, they help mitigate the NO produced by the immune system of the host, and aid survival during fluctuating concentrations concentrations of oxygen. FDPs have a mosaic structure, being predicted to contain multiple extra domains. Clostridium difficile, a threatening human pathogen, encodes two FDPs: one with the two canonical domains, and another with a larger polypeptide chain of 843 amino acids, CD1623, with two extra domains, predicted to be a short-rubredoxin-like and an NAD(P)H:rubredoxin oxidoreductase. This multi-domain protein is the most complex FDP characterized thus far. Each of the predicted domains was characterized and the presence of the predicted cofactors confirmed by biochemical and spectroscopic analysis. Results show that this protein operates as a standalone FDP, receiving electrons directly from NADH, and reducing oxygen to water, precluding the need for extra partners. CD1623 displayed negligible NO reductase activity, and is thus considered an oxygen selective FDP, that may contribute to the survival of C. difficile in the human gut and in the environment.

The σB signalling activation pathway in the enteropathogen Clostridioides difficile

“…3C). However, the expression of CD1474, CD0176 and CD1623 encoding proteins likely involved in O 2 or ROS detoxification pathways (Kint et al, 2017;Folgosa et al, 2018) decreased in the ΔrsbV mutant compared to the wild-type strain ( Fig. 3C).…”

“…Moreover, while sigB is drastically overexpressed, the expression of several C. difficile σ B -target genes transcribed from a σ B -dependent promoter only moderately increased. Indeed, the expression of CD1623 and CD1157 encoding flavodiiron proteins likely involved in the oxidative stress response (Folgosa et al, 2018) as well as CD1474 and CD0176, encoding a reverse rubrerythrin and an NAD/FAD oxidoreductase, respectively, was upregulated only up to fivefold (Fig. 2C).…”

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle

“…Flavodiiron proteins belong to a singular family of oxygen and/or nitric oxide reductases widespread in prokaryote and eukaryote organisms. This protein family has been classified into eight classes (A to H) according to the presence of modular domains [65,66]. Class A FDPs are the most dominant class in the family and only contain the two core domains (Fe-Fe and FMN) [65,66].…”

“…This protein family has been classified into eight classes (A to H) according to the presence of modular domains [65,66]. Class A FDPs are the most dominant class in the family and only contain the two core domains (Fe-Fe and FMN) [65,66]. Some examples for this class A show a preference for O 2 as revealed for the protozoal and archaeal enzyme [67][68][69][70], or they may have a dual activity toward NO and O 2 as in M. thermoacetica or D. gigas [71,72].…”

“…Some examples for this class A show a preference for O 2 as revealed for the protozoal and archaeal enzyme [67][68][69][70], or they may have a dual activity toward NO and O 2 as in M. thermoacetica or D. gigas [71,72]. Class B FDPs contain an additional C-terminal rubredoxin domain and, because of that, were named flavorubredoxins (FIRD) or NORV (from the encoding gene in E. coli) [65]. In E. coli, FLVB/FIRD/NORV was shown to have significant preference for NO over O 2 and confers resistance to NO under anaerobic conditions [73,74].…”