Reaction Mechanism and Regulation of Mammalian Thioredoxin/Glutathione Reductase

Abstract

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.

Mapping the Catalytic Cycle of Schistosoma mansoni Thioredoxin Glutathione Reductase by X-ray Crystallography

“…In this study, we solved several structures of this enzyme in complex with some of its substrates, and it would be highly desirable to assign these structures to the reaction intermediates. A partial description of the enzymatic cycle has been proposed for mouse TGR, mainly based on the better characterized cycle of TRs and on a threedimensional model of mouse TGR (18,35). Our structures fit the known or presumed catalytic intermediates of mouse TGR as follows; Structure 1 clearly corresponds to a unique species of SmTGR (the oxidized one).…”

“…The catalytic mechanism of TGR has not been fully elucidated, although an hypothesis has been published for the mouse enzyme (18). Functional analysis performed on SmTGR and on different TGRs proved that the penultimate Sec is essential for the reduction of thioredoxin and the glutaredoxin domain (6,12,15).…”

“…Electron transfer in and out of the TR domain of SmTGR. The reaction scheme has been drawn following the experimental evidence reported in this work and elsewhere (18 functionally relevant movements of the C terminus during the redox cycle of the enzyme.…”

The proteasome and its role in the degradation of oxidized proteins

“…The first level is formed by low-molecular weight antioxidants, the second level includes enzymatic antioxidative defence, and the third level enables cells to repair oxidatively modified proteins. Modified cystein residues (sulfenes, sulfines, or sulfones) (22,23) and methionine sulfoxides via the methionine-sulfoxidreductase-system (24,25) can be repaired to restore the unmodified form of the damaged protein. If any other residue is oxidized, the protein is irreversibly damaged and should be degraded.…”

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

“…Possessing an N-terminal Grx domain in addition to the C-terminal selenocysteine-containing redox center, this TrxR-GR chimeric enzyme exerts TrxR, GR and Grx activities [11].…”