Cysteine oxidation as a regulatory mechanism of Arabidopsis plastidial NAD‐dependent malate dehydrogenase

Abstract

Malate dehydrogenases (MDHs) catalyze a reversible NAD(P)‐dependent‐oxidoreductase reaction that plays an important role in central metabolism and redox homeostasis of plant cells. Recent studies suggest a moonlighting function of plastidial NAD‐dependent MDH (plNAD‐MDH; EC 1.1.1.37) in plastid biogenesis, independent of its enzyme activity. In this study, redox effects on activity and conformation of recombinant plNAD‐MDH from Arabidopsis thaliana were investigated. We show that reduced plNAD‐MDH is active while it is inhibited upon oxidation. Interestingly, the presence of its cofactors NAD+ and NADH could prevent oxidative inhibition of plNAD‐MDH. In addition, a conformational change upon oxidation could be observed via non‐reducing SDS‐PAGE. Both effects, its inhibition and conformational change, were reversible by re‐reduction. Further investigation of single cysteine substitutions and mass spectrometry revealed that oxidation of plNAD‐MDH leads to oxidation of all four cysteine residues. However, cysteine oxidation of C129 leads to inhibition of plNAD‐MDH activity and oxidation of C147 induces its conformational change. In contrast, oxidation of C190 and C333 does not affect plNAD‐MDH activity or structure. Our results demonstrate that plNAD‐MDH activity can be reversibly inhibited, but not inactivated, by cysteine oxidation and might be co‐regulated by the availability of its cofactors in vivo.