Biochemical Function, Molecular Structure and Evolution of an Atypical Thioredoxin Reductase from Desulfovibrio vulgaris.

Odile Valette,Alain Dolla,Christine Cavazza,Gaël Brasseur,Tam T T Tran,Elodie Caudeville,Emmanuel Talla,Laetitia Pieulle

doi:10.3389/fmicb.2017.01855

Abstract

Thioredoxin reductase (TR) regulates the intracellular redox environment by reducing thioredoxin (Trx). In anaerobes, recent findings indicate that the Trx redox network is implicated in the global redox regulation of metabolism but also actively participates in protecting cells against O2. In the anaerobe Desulfovibrio vulgaris Hildenborough (DvH), there is an intriguing redundancy of the Trx system which includes a classical system using NADPH as electron source, a non-canonical system using NADH and an isolated TR (DvTRi). The functionality of DvTRi was questioned due to its lack of reactivity with DvTrxs. Structural analysis shows that DvTRi is a NAD(P)H-independent TR but its reducer needs still to be identified. Moreover, DvTRi reduced by an artificial electron source is able to reduce in turn DvTrx1 and complexation experiments demonstrate a direct interaction between DvTRi and DvTrx1. The deletion mutant tri exhibits a higher sensitivity to disulfide stress and the gene tri is upregulated by O2 exposure. Having DvTRi in addition to DvTR1 as electron source for reducing DvTrx1 must be an asset to combat oxidative stress. Large-scale phylogenomics analyses show that TRi homologs are confined within the anaerobes. All TRi proteins displayed a conserved TQ/NGK motif instead of the HRRD motif, which is selective for the binding of the 2′-phosphate group of NADPH. The evolutionary history of TRs indicates that tr1 is the common gene ancestor in prokaryotes, affected by both gene duplications and horizontal gene events, therefore leading to the appearance of TRi through subfunctionalization over the evolutionary time.

Highlights

The maintenance of a proper disulfide state in the extracellular or intracellular environment is crucial for cell survival
A rapid reduction of the bound flavin adenine dinucleotide (FAD) represented by a dramatic decrease in the absorbance, was observed when an excess of dithionite was added
As previously demonstrated (Pieulle et al, 2011), we found that DvTR1 and DvTR3 were efficiently reduced by their specific reductants (Supplementary Figures S3C,D)

Summary

Introduction

The maintenance of a proper disulfide state in the extracellular or intracellular environment is crucial for cell survival. As reductants of intracellular disulfides, the principal protein systems that control the thiol/disulfide redox balance in the cytoplasm are the NADPH-dependentTR/Trx system and the glutathione reductase/glutathione/glutaredoxin pathway. Trx is a small redox-active protein (∼12 kDa), which has a conserved CxxC catalytic site that undergoes reversible oxidation/reduction of both cysteine residues whereas TR is a member of the flavoprotein family of pyridine nucleotide disulfide oxidoreductases and functions in a homodimeric form. In the FO conformation, the flavin and the disulfide were juxtaposed for flavin oxidation and the redox-active disulfide pair is in a buried state, forbidding direct interaction with Trx (Lennon et al, 2000). The FR conformation is required for the reduction of the large protein substrate Trx by the TR dithiol

Methods

Results

Conclusion