Functional metagenomics of the thioredoxin superfamily

Abstract

Environmental sequence data of microbial communities now makes up the majority of public genomic information. The assignment of a function to sequences from these metagenomic sources is challenging because organisms associated with the data are often uncharacterized and not cultivable. To overcome these challenges, we created a rationally designed expression library of metagenomic proteins covering the sequence space of the thioredoxin superfamily. This library of 100 individual proteins represents more than 22,000 thioredoxins found in the Global Ocean Sampling data set. We screened this library for the functional rescue of Escherichia coli mutants lacking the thioredoxin-type reductase (Δ trxA ), isomerase (Δ dsbC ), or oxidase (Δ dsbA ). We were able to assign functions to more than a quarter of our representative proteins. The in vivo function of a given representative could not be predicted by phylogenetic relation but did correlate with the predicted isoelectric surface potential of the protein. Selected proteins were then purified, and we determined their activity using a standard insulin reduction assay and measured their redox potential. An unexpected gel shift of protein E5 during the redox potential determination revealed a redox cycle distinct from that of typical thioredoxin-superfamily oxidoreductases. Instead of the intramolecular disulfide bond formation typical for thioredoxins, this protein forms an intermolecular disulfide between the attacking cysteines of two separate subunits during its catalytic cycle. Our functional metagenomic approach proved not only useful to assign in vivo functions to representatives of thousands of proteins but also uncovered a novel reaction mechanism in a seemingly well-known protein superfamily.

Auranofin induces disulfide bond-mimicking S-Au-S bonds in protein thiol pairs

“…The trxA gene from B. subtilis encoding thioredoxin was amplified from B. subtilis 168 genomic DNA via PCR with the primers LQB001_for and LQB002_rev. The PCR product was digested with NdeI and BamHI and cloned into the pOE vector (Nilewski et al, 2021), yielding the plasmid pLQB, containing trxA fused to an N-terminal His 6 -tag and a TEV protease cutting site.…”

“…The insulin reduction assay was performed as described by Nilewski et al . (Nilewski et al, 2021). Briefly, FITC-labeled insulin reduction was monitored fluorometrically with a fluorescence spectrophotometer FP-8500 (Jasco) equipped with a Peltier-thermo cell holder EHC-813.…”

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding inEscherichia coli

“…For construction of the pPT plasmid enabling periplasmic targeting, the torA sequence was PCR amplified using appropriate primers (Supplementary table S3) from genomic E. coli DNA (strain MC4100) and cloned into the pCC plasmid (Nilewski et al, 2021) using the introduced Nde I 3’ and 5’ restriction sites. The Nde I restriction site upstream of torA was then removed by QuickChange mutagenesis (Agilent, Waldbronn, Germany) according to the manufacturer’s protocol using primers QC- Nde I-fw and QC- Nde I-rv (Supplementary table S3) resulting in the pPT plasmid.…”

Experimental Approaches for Investigating Disulfide-Based Redox Relays in Cells

“…Despite decades of study, the distinct biological substrate specificities of functional glutaredoxins and thioredoxins remain unresolved in many species, with our understanding largely being pieced together from in vitro work or biological studies focused on a limited number of substrates, as opposed to more systematic, comprehensive approaches. Similarly, the redox partners and biological roles of many members of the larger thioredoxin superfamily, along with other predicted thiol-dependent oxidoreductases, remain to be studied, ,, as do the systems that carry out disulfide formation and rearrangement in the exoplasm of many Gram-positive bacteria. , Combiningadvances in proteomic technologies, biorthogonal chemistry, and genome manipulation with the methods described herein, the future holds great promise for more fully understanding each of these and other underexplored disulfide-based redox relays.…”