Salmonella enterica BcfH Is a Trimeric Thioredoxin-Like Bifunctional Enzyme with Both Thiol Oxidase and Disulfide Isomerase Activities

Abstract

Aims: Thioredoxin (TRX)-fold proteins are ubiquitous in nature. This redox scaffold has evolved to enable a variety of functions, including redox regulation, protein folding, and oxidative stress defense. In bacteria, the TRX-like disulfide bond (Dsb) family mediates the oxidative folding of multiple proteins required for fitness and pathogenic potential. Conventionally, Dsb proteins have specific redox functions with monomeric and dimeric Dsbs exclusively catalyzing thiol oxidation and disulfide isomerization, respectively. This contrasts with the eukaryotic disulfide forming machinery where the modular TRX protein disulfide isomerase (PDI) mediates thiol oxidation and disulfide reshuffling. In this study, we identified and structurally and biochemically characterized a novel Dsb-like protein from Salmonella enterica termed bovine colonization factor protein H (BcfH) and defined its role in virulence. Results: In the conserved bovine colonization factor (bcf) fimbrial operon, the Dsb-like enzyme BcfH forms a trimeric structure, exceptionally uncommon among the large and evolutionary conserved TRX superfamily. This protein also displays very unusual catalytic redox centers, including an unwound a-helix holding the redox active site and a trans-proline instead of the conserved cis-proline active site loop. Remarkably, BcfH displays both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis. Innovation and Conclusion: Typically, oligomerization of bacterial Dsb proteins modulates their redox function, with monomeric and dimeric Dsbs mediating thiol oxidation and disulfide isomerization, respectively. This study demonstrates a further structural and functional malleability in the TRX-fold protein family. BcfH trimeric architecture and unconventional catalytic sites permit multiple redox functions emulating in bacteria the eukaryotic PDI dual oxidoreductase activity. Antioxid. Redox Signal. 35,[21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39]

The suppressor of copper sensitivity protein C from Caulobacter crescentus is a trimeric disulfide isomerase that binds copper(I) with subpicomolar affinity

“…Moreover, the putative N-terminal oligomerization domains (50 residues after the signal sequence of both PmScsC and CcScsC) share more than 30% sequence identity. Comparison of the N-terminal sequence of CcScsC with those of other bacterial oxidoreductases that have been reported to be trimeric [S. Typhimurium BcfH (StBcfH;Subedi et al, 2021) and Wolbachia pipientis -DsbA2 (WpDsbA2; Walden et al, 2019)] further highlights similarities between the trimeric proteins and CcScsC (Fig. 1c).…”

“…It has been suggested that this segment allows the three catalytic domains of PmScsC to explore a wide range of conformations during the refolding of bound misfolded substrates (Furlong et al, 2017(Furlong et al, , 2019. Trimeric StBcfH adopts at least two different conformations (found in the crystal asymmetric unit), and like eukaryotic protein disulfide isomerases (Soares Moretti & Martins Laurindo, 2017) StBcfH is both a dithiol oxidase and a disulfide isomerase (Subedi et al, 2021). For trimeric WpDsbA2, SAXS data analysis suggests that the N-terminal trimerization domain is rigid and may contribute to disulfide isomerase activity simply by bringing three DsbA-like domains into close proximity (Walden et al, 2019).…”

“…Swarming-motility assays under NaCl-free conditions were performed as described previously (Subedi et al, 2021). Briefly, overnight cultures of P. mirabilis strains grown in the presence of 100 mM IPTG and 17 mg ml À1 chloramphenicol in NaCl-free LB were streaked onto NaCl-free LB agar containing 100 mM IPTG and 17 mg ml À1 chloramphenicol.…”

Structural bioinformatic analysis of DsbA proteins and their pathogenicity associated substrates

“…Examples include uropathogenic E. coli (UPEC) strain CFT073 that possesses a canonical DsbA and an accessory DsbA homologue DsbL [19] , [54] or the widely distributed facultative anaerobe Shewanella oneidensis , which has a complex DSB machinery with four DsbA (DsbA1-4) and two DsbB (DsbB1-2) homologues [59] . Similarly, Salmonella enterica ( Se ) encodes four DsbA-like proteins [41] , namely DsbA, DsbL, SrgA [42] , a virulence plasmid-encoded DsbA-like protein, and ScsC [60] , [61] , a DsbA-like protein that protects against copper toxicity [57] , [62] . These Salmonella DsbA paralogues were found to share low sequence identity along with significant differences in their surface features and redox properties [41] .…”

“…Interestingly, BcfH has been shown to form a trimeric structure, exceptionally uncommon among the thioredoxin superfamily members. Additionally, BcfH has both thiol oxidase and disulfide isomerase activities contributing to Salmonella fimbrial biogenesis [57] .…”