ENGINEERING A DOMAIN-LOCKING DISULFIDE INTO A BACTERIAL MALATE-DEHYDROGENASE PRODUCES A REDOX-SENSITIVE ENZYME

Light-dependent reduction of cystine disulfide bonds results in activa tion of several of the enzymes of photosynthetic carbon metabolism wit hin the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenas e, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoh eptulosebisphosphatase, and identified cysteines in each enzyme that m ight be expected to form inactivating disulfide bonds (Li, D., F. J. S tevens, M. Schiffer, and L. E. Anderson, 1994. Biophys. J. 67:29-35). We have now converted two residues in the Escherichia coli NAD-linked malate dehydrogenase to cysteines and produced a redox-sensitive enzym e. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast de hydrogenase. This result is completely consistent with our proposed me chanism.