Functional metagenomics of the thioredoxin superfamily.
Catalysis
Cysteine
/ chemistry
Environmental Monitoring
Escherichia coli
/ genetics
Glutaredoxins
/ chemistry
Metagenomics
Multigene Family
/ genetics
Oceans and Seas
Oxidation-Reduction
Phylogeny
Protein Disulfide-Isomerases
/ chemistry
Thioredoxin-Disulfide Reductase
/ chemistry
Thioredoxins
/ chemistry
DsbA
DsbC
Escherichia coli (E. coli)
TrxA
oxidase
protein disulfide isomerase
reductase
thiol
thiol-disulfide oxidoreductase
thioredoxin
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
Historique:
received:
09
10
2020
revised:
18
12
2020
accepted:
24
12
2020
pubmed:
29
12
2020
medline:
25
8
2021
entrez:
28
12
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33361108
pii: S0021-9258(21)00013-2
doi: 10.1074/jbc.RA120.016350
pmc: PMC7949104
pii:
doi:
Substances chimiques
Glutaredoxins
0
Thioredoxins
52500-60-4
Thioredoxin-Disulfide Reductase
EC 1.8.1.9
Protein Disulfide-Isomerases
EC 5.3.4.1
Cysteine
K848JZ4886
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
100247Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
Références
Proteomics. 2010 Feb;10(4):771-84
pubmed: 19953543
Biochemistry. 1995 Apr 18;34(15):5075-89
pubmed: 7536035
J Mol Graph. 1996 Feb;14(1):33-8, 27-8
pubmed: 8744570
Nat Commun. 2014 Dec 17;5:5804
pubmed: 25517874
Nucleic Acids Res. 1988 Sep 12;16(17):8726
pubmed: 3047688
Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5
pubmed: 10829079
Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W526-31
pubmed: 15980527
J Bacteriol. 1974 Sep;119(3):736-47
pubmed: 4604283
Structure. 1995 Mar 15;3(3):245-50
pubmed: 7788290
J Biol Chem. 2013 Jul 5;288(27):19698-714
pubmed: 23696645
Bioinformatics. 2006 Jul 1;22(13):1658-9
pubmed: 16731699
Structure. 2001 Nov;9(11):1071-81
pubmed: 11709171
Free Radic Biol Med. 2001 Jun 1;30(11):1191-212
pubmed: 11368918
J Comput Chem. 2004 Oct;25(13):1605-12
pubmed: 15264254
Mol Biol Evol. 2013 Apr;30(4):772-80
pubmed: 23329690
PLoS Biol. 2007 Mar;5(3):e16
pubmed: 17355171
J Biol Chem. 1988 Oct 15;263(29):14684-9
pubmed: 3049584
J Biol Chem. 1980 Nov 10;255(21):10261-5
pubmed: 7000775
Eur J Biochem. 1983 Oct 17;136(1):223-32
pubmed: 6352262
Nucleic Acids Res. 2007 Jul;35(Web Server issue):W522-5
pubmed: 17488841
J Bacteriol. 1999 Mar;181(5):1375-9
pubmed: 10049365
Nat Methods. 2011 Sep 29;8(10):785-6
pubmed: 21959131
Protein Sci. 2004 Oct;13(10):2744-52
pubmed: 15340164
J Virol. 2002 Oct;76(20):10245-55
pubmed: 12239300
Protein Sci. 1993 May;2(5):717-26
pubmed: 8495194
Biol Chem. 2019 Apr 24;400(5):575-587
pubmed: 30367780
Nat Struct Mol Biol. 2011 May;18(5):592-6
pubmed: 21460845
Proc Natl Acad Sci U S A. 1975 Jun;72(6):2305-9
pubmed: 1094461
Nat Struct Biol. 1998 Jun;5(6):476-83
pubmed: 9628486
J Biol Chem. 2002 Mar 29;277(13):10861-8
pubmed: 11741965
Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11224-9
pubmed: 11553771
EMBO J. 1994 Apr 15;13(8):2007-12
pubmed: 8168497
Nucleic Acids Res. 2014 Jan;42(Database issue):D222-30
pubmed: 24288371
Proc Natl Acad Sci U S A. 2001 Aug 28;98(18):10037-41
pubmed: 11517324
PLoS Comput Biol. 2011 Oct;7(10):e1002195
pubmed: 22039361
Bioinformatics. 2006 Jan 15;22(2):195-201
pubmed: 16301204
J Biol Chem. 1991 Mar 5;266(7):4056-66
pubmed: 1999401
J Bacteriol. 2006 Jun;188(12):4264-70
pubmed: 16740933
J Biol Chem. 1979 Oct 10;254(19):9627-32
pubmed: 385588
Nucleic Acids Res. 2002 Apr 1;30(7):1575-84
pubmed: 11917018
J Bacteriol. 2005 Oct;187(19):6770-8
pubmed: 16166540
J Biol Chem. 1970 May 10;245(9):2371-4
pubmed: 4392601
Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1038-42
pubmed: 8430071
Nat Methods. 2012 Jul;9(7):671-5
pubmed: 22930834
Microbiol Rev. 1983 Mar;47(1):1-45
pubmed: 6343825
J Biol Chem. 2004 Mar 26;279(13):12967-73
pubmed: 14726535
Proc Natl Acad Sci U S A. 1993 Feb 1;90(3):1043-7
pubmed: 8503954
J Mol Biol. 1990 Oct 5;215(3):403-10
pubmed: 2231712
Antioxid Redox Signal. 2010 Oct;13(8):1205-16
pubmed: 20136512