Joris Messens

Thiol-based Catalysis and Redox Regulation

A number of cellular oxidation and reduction pathways rely on the relay of electrons between pairs of cysteines and seem to be controlled by the specificity of intra- and inter-protein interactions. Many enzymatic pathways describe a conserved thiol-disulfide exchange mechanism to transfer disulfide bonds between separate components of the cellular redox systems. In addition to these inter-protein transfer events, eukaryotic and prokaryotic pathways share mechanisms for disulfide-bond transfer between several pairs of cysteines within a single protein. 

For our research work, we focus on enzymes in which cysteines lie at the heart of the catalysis mechanism and on the oxidative folding process by which a protein recovers both its native disulfide bonds and its native structure.

We unravelled the reaction mechanism of pI258 arsenate reductase (ArsC) from S. aureus, a redox enzyme with four cysteines. Biochemistry, quantum chemistry, kinetic studies, NMR and X-ray crystallography have provided insight into the reduction mechanism of ArsC. This redox-enzyme combines a phosphatase-like nucleophilic displacement reaction with a unique intramolecular disulfide bond cascade. Within this cascade the formation of a disulfide bond triggers a reversible conformational switch that transfers the oxidative equivalents to the surface of the protein. 

Recently, we identified the first enzymes, which use mycothiol and mycoredoxin in a thiol/disulfide redox cascade. The enzymes are two arsenate reductases from Corynebacterium glutamicum (Cg_ArsC1 and Cg_ArsC2), which play a key role in the defence against arsenate. In vivo knockouts showed that the genes for Cg_ArsC1 and Cg_ArsC2 and those of the enzymes of the mycothiol biosynthesis pathway confer arsenate resistance. With steady-state kinetics, arsenite analysis, and theoretical reactivity analysis, we unravelled the catalytic mechanism for the reduction of arsenate to arsenite in C. glutamicum. The active site thiolate in Cg_ArsCs facilitates adduct formation between arsenate and mycothiol. Mycoredoxin - a redox enzyme for which the function was never shown before - reduces the thiol-arseno bond, forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that on its turn is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of ~5 M^-1s^-1, typically for a thiol/disulfide cascade with nucleophiles on three different molecules. With the in vitro reconstitution of this novel electron transfer pathway, we have paved the way for the study of redox mechanisms in pathogenic actinobacteria, like Mycobacterium tuberculosis.

Another enzyme that we have been studying is the ubiquitous reductant thioredoxin (Trx). We showed that Trx is able to discriminate between the folds of reduced and oxidized substrate. Trx itself gets its specificity from its conserved active site WCGPC motif, in which the proline residue determines the driving force to reduce substrate proteins.

For oxidative protein folding, cells possess protein-folding catalysts to ensure that the correct pairs of cysteine residues are joined. In general view, the folding of proteins with nonconsecutive disulfides requires protein disulfide isomerases. Through studies of wild type and disulfide mutants of RNase I as substrates for the Dsb oxidoreductases in E. coli, we showed that a protein with a nonconsecutive disulfide may in vivo as well as in vitro fold to its proper structure with DsbA alone and that the dependence on DsbC is linked to the redox state of the environment. The kinetics of the oxidative folding process and the structures of redox proteins caught in action as a mixed disulfide complex present new challenges for the future. Further, in order to reach for the disulfide rich, high hanging fruits of structural biology, we are developing in vitro folding technology using cell-free expression technology, called Smooth Funnel technology. With this SF-technology we will efficiently fold recombinant proteins.

Brussels Center for Redox Biology