Arsenate Reductase, Mycothiol, and Mycoredoxin Concert Thiol/Disulfide Exchange

Efrén Ordóñez,Karolien Van Belle,Goedele Roos,Sandra De Galan,Michal Letek,Jose A Gil,Lode Wyns,Luis M Mateos,Joris Messens

doi:10.1074/jbc.m900877200

Abstract

We identified the first enzymes that 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 defense 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 unraveled 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 and forms arsenite and a mycothiol-mycoredoxin mixed disulfide. A second molecule of mycothiol recycles mycoredoxin and forms mycothione that, in its turn, is reduced by the NADPH-dependent mycothione reductase. Cg_ArsCs show a low specificity constant of approximately 5 m(-1) s(-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 actinobacteria.

Highlights

Ferent thiolate nucleophiles that function as a redox cascade
Most of the cellular arsenate resistance is associated with the presence of cellular arsenate reductases (ArsC/Acr2p)
In C. glutamicum, we have found a completely new enzymatic mechanism for the reduction of arsenate in which the electron transfer is coupled to mycothiol (MSH) and mycoredoxin1 (Mrx1) (Fig. 6)

Summary

EXPERIMENTAL PROCEDURES

Knockouts Involved in Mycothiol Biosynthesis—MshB, MshC, and MshD mutant strains from C. glutamicum were described previously [22]. Oxidized Mrx was prepared by the addition of a 10-fold molar excess of diamide, followed by size exclusion chromatography on Superdex HR in 50 mM Hepes, pH 8.0, 150 mM NaCl. Arsenite Analysis—All components (or an experimentally designed selection) were mixed in 20 mM Tris/HCl, pH 6.5, to obtain 250 ␮M NADPH, 3 ␮M MTR, 10 ␮M Mrx1, 0.47 mM MSH, 200 nM Cg_ArsCs, and 100 mM As(V) (varying concentrations) incubated for different times at 37 °C. None of the arsC genes increased the survival of the WC3110 strain in arsenate (data not shown) This might indicate that the genes for arsenate reductases in C. glutamicum have evolved to become hostspecific enzymes, which depend on arsenate or sulfur atom of HAsO42Ϫ, CH3S-AsO32Ϫ, and CH3SHAsO3Ϫ can be extracted from the difference in the local softness between the reacting partners (hard and soft acids and bases principle) [26]. The local softness was calculated as described [27]

RESULTS

DISCUSSION