Functional metagenomics of the thioredoxin superfamily
| Autor: | Lars I. Leichert, Anne F Baumann, Christopher Horst Lillig, Natalie Lupilov, Witali Kusnezow, Anna Kusnezowa, Mehmet Berkmen, Marharyta Varatnitskaya, Manuela Gellert, Martin Eisenacher, Markus-Hermann Koch, Thorsten Masuch, Sebastian Nilewski |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Isomerase AMS 4-acetamido-4'-maleimidylstilbene-2 2'-disulfonic acid Biochemistry Thioredoxins thiol-disulfide oxidoreductase Protein disulfide-isomerase Phylogeny Escherichia coli (E. coli) biology Chemistry oxidase thiol pOE plasmid for overexpression protein disulfide isomerase Multigene Family PNK polynucleotide kinase PAPS 3'-phosphoadenosine-5'-phosphosulfate Sequence space (evolution) Thioredoxin Oxidation-Reduction Environmental Monitoring Research Article PDIs protein disulfide isomerases Thioredoxin-Disulfide Reductase Oceans and Seas Protein Disulfide-Isomerases TrxA pPC plasmid for periplasmic complementation Computational biology TCA trichloroacetic acid Catalysis TEV tobacco etch virus 03 medical and health sciences GOS Global Ocean Sampling pCC plasmid for cytoplasmic complementation vtPA truncated version of human tissue plasminogen activator Escherichia coli Cysteine Molecular Biology Glutaredoxins 030102 biochemistry & molecular biology DsbA Ni–NTA nickel–nitrilotriacetic acid Cell Biology thioredoxin Protein superfamily DsbC 030104 developmental biology Metagenomics reductase biology.protein CXXC Cys-X-X-Cys Function (biology) |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | 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 (ΔtrxA), isomerase (ΔdsbC), or oxidase (ΔdsbA). 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. |
| Databáze: | OpenAIRE |
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