Multi-domain flavodiiron proteins from Trichomonas vaginalis

BIROCCO, F; GUERRERO, SA; IGLESIAS, AA; ARIAS, DG

Reunión virtual

Workshop; Tercer Encuentro & Primer Workshop de la Red Argentina de Tecnología Enzimática (Red TEz); 2021

Red Argentina de Tecnología Enzimática (Red TEz)

Trichomoniasis,caused by the extracellular parasite Trichomonasvaginalis, is the most common non-viralsexually transmitted disease in the world. Instead of mitochondria, T. vaginalis has hydrogenosomes thatproduce H2 as an end product of anaerobic energy metabolism. Coreenzymes of the hydrogenosome metabolism, such as pyruvate-ferredoxinoxidoreductase and Fe?Fe hydrogenases, are highly oxygen-sensitive. However,the parasite typically experiences oxygen stress in its natural environment,during the host transmission and hence must possess mechanisms to avoidinactivation of oxygen-sensitive enzymes and to remove reactive oxygen species.Some decades ago, it was affirmed that cytosolic NADH and NADPH oxidases areamong the parasite?s most important O2 scavenging enzymes, andrecently, a single-domain flavodiiron protein (FDP) with hydrogenosomelocalization was studied. The single-domain FDPs are enzymes with a minimal core of two domains: a metallo-β-lactamase like harboring a diiron center, and aflavodoxin, FMN containing domains. FDPs are O2 or NO reducingenzymes; for many pathogens, they help mitigate the NO produced by the immunesystem of the host, and aid survival during fluctuating concentrations ofoxygen. FDPs have a mosaic structure, being predicted to contain multiple extradomains. In this work, we present the functional characterizationof two isoforms of multi-domain FDP (TvNROR‑FDP1and TvNROR-FDP2), which belong to aclass-F, protein with a modular arrangement. In silico analysis showed that the proteins have two FDP canonical domainsand two extra domains, predicted to be a short rubredoxin-like and aNAD(P)H:rubredoxin oxidoreductase. TvNROR‑FDP1and TvNROR-FDP2 were obtained byrecombinant expression in Escherichia coli andpurified proteins presented a dimeric structure by gel filtration chromatography. Theability to ligate prosthetic groups by the recombinant enzymes was evaluated byUV-Vis spectroscopy, TLC experiments, and metal quantification, showingevidence that TvNROR-FDPs conjugate FMN and/or FAD and iron atoms.Biochemicalassays showed that both of them wereable to catalyse the in vitro NAD(P)H-dependent O2 reduction.TvNROR-FDP1, but not TvNROR-FDP2, exhibited the NADH-dependent O2partial-reduction to H2O2. ForO2 reductase activity, although the pH‑dependent activity profile resultedsimilar for both enzymes, it was observed profile coenzyme dependence. Inaddition, the inactivation by DPI-Cl showed that flavin moiety is essential forthe TvNROR-FDP activity. TvNROR-FDP1 showed the capability ofelectron transferring to one and two-electron-acceptor compounds, giving riseto diaphorase activity. However, the enzymes showed low activity for exogenousrubredoxin reduction. Basal expression was detected in T. vaginalis trophozoitesby western blot experiment and their subcellular localization pattern revealedcytoplasmatic patterns. Altogether, the resultssuggest that TvNROR-FDP1/2could be involved in oxidative stress protection in the parasite. To the best of ourknowledge, this is the first characterization ofclass-F FDPs from a eukaryotic organism. Grantedby ANPCyT (PICT2016-1778 and PICT2017-2268).