Study of the Thiol/Disulfide Redox Systems of the Anaerobe Desulfovibrio vulgaris Points Out Pyruvate:Ferredoxin Oxidoreductase as a New Target for Thioredoxin 1

Abstract

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balancecontrolling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K m value for Trx1 of 8.9 M. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K m for NADPH, 743 M; K m for NADH, 5.6 M), and Trx3 was unable to reduce insulin. The K m value of TR3 for Trx3 was 1.12 M. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1 C33S as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms from all three domains of life (1-3). To combat this problem, anaerobes have evolved multifaceted strategies to manage the deleterious effects of oxygen (O 2 ) exposure. In this regard, these organisms demonstrate varying degrees of tolerance to O 2 , ranging from the extremely sensitive methanogens, which typically are inhibited by only a few ppm of O 2 (4), to the much more aerotolerant Bacteroides (5) or sulfate-reducing Desulfovibrio species (6, 7). The extreme aerotolerance of these anaerobes can be related to their way of life. Abundance and metabolic activity of sulfate reducers in oxic zones of numerous biotopes (reviewed in Ref. 8) are frequently evaluated as higher than the ones found in neighboring anoxic zones. From the last decade, studies have uncovered original and complex adaptative strategies by which sulfate reducers seek to minimize the damage induced by oxidative conditions (8). One example is superoxide reductase, which is specific to anaerobes and scavenges superoxide ions by reduction. Although this enzyme is broadly distributed in sulfatereducing bacteria (9 -12), more species-specific mechanisms are also found as the disulfide bond-mediated protection of the pyruvate:ferredoxin oxidoreductase (PFOR) 3 (13). In Desulfovibrio africanus, this enzyme, which cont...

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…However, their relationships with the phylogenetically nearest neighbors suggested that the TrxRs of the Unknown I group are likely to be NTRs. The T. maritima TrxR that can use both NADH and NADPH (16) belonged to an NTR group containing bona fide enzymes from D. vulgaris (D.vulgaris_2, accession number AAS94860) (6) and Methanosarcina acetivorans (accession number, AAM04784) (9).…”

“…For example, the isolation of the putative Trx targets via Trxaffinity chromatography and the results from activation assays with select purified and deactivated enzymes have established that in Chlorobaculum tepidum, a green sulfur bacterium and an anaerobic phototroph, the thioredoxin system implements post-translational control on the tricarboxylic acid or the TCA cycle and sulfur metabolism and assists in the defense against oxidative stress (7). An analysis of mutant phenotypes and a gene expression study have revealed the critical roles of Trxs and TrxRs in the redox homeostasis and survival during oxidative stress in Bacteroides fragilis and Desulfovibrio vulgaris Hildenborough, respectively (6,14). Work with Clostridium pasteurianum, an anaerobic fermentative bacterium, has revealed a ferredoxin-dependent flavin containing TrxR (8).…”

“…However, it has also been investigated significantly in the anaerobic organisms (6 -24) focusing on both the characteristics of the Trxs and TrxRs (6,8,9,(11)(12)(13)(15)(16)(17)(18)(19)(20)(21)(22)(23) and their physiological roles (6,7,10,14,20). For example, the isolation of the putative Trx targets via Trxaffinity chromatography and the results from activation assays with select purified and deactivated enzymes have established that in Chlorobaculum tepidum, a green sulfur bacterium and an anaerobic phototroph, the thioredoxin system implements post-translational control on the tricarboxylic acid or the TCA cycle and sulfur metabolism and assists in the defense against oxidative stress (7).…”

Structural and Mechanistic Insights into Unusual Thiol Disulfide Oxidoreductase

“…In Desulfovibrio vulgaris Hildenborough, a Gram-negative sulfate-reducing bacteria, two cytoplasmic thioredoxin systems have been identified (14). The Trx1/TR1 system contains the ubiquitous thioredoxin with the active site consensus sequence WCGPC.…”

“…The Trx1/TR1 system contains the ubiquitous thioredoxin with the active site consensus sequence WCGPC. The second system contains an atypical thioredoxin with a CPHC sequence at the active site identical to the DsbA motif and an unconventional thioredoxin reductase that uses preferentially NADH (14). The presence of these atypical proteins being restricted to Desulfovibrio organisms, they have been named desulfothioredoxin (Dtrx) and desulfothioredoxin reductase (15).…”