Engineering a domain-locking disulfide into a bacterial malate dehydrogenase produces a redox-sensitive enzyme.
Light-dependent reduction of cystine disulfide bonds results in activation of several of the enzymes of photosynthetic carbon metabolism within the chloroplast. We have modeled the tertiary structure of four of these light-activated enzymes, namely NADP-linked malate dehydrogenase, glyceraldehyde-3-P dehydrogenase, fructosebisphosphatase, and sedoheptulosebisphosphatase, and identified cysteines in each enzyme that be expected to form inactivating disulfide bonds (Li, D., F. J. Stevens, 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 enzyme. Oxidation of domain-locking cysteine residues in the mutant enzyme clearly mimics dark inactivation of the redox-sensitive chloroplast dehydrogenase. This result is completely consistent with our proposed mechanism.
Muslin
E H
EH
Department of Biological Sciences, University of Illinois at Chicago 60607, USA.
Li
D
D
Stevens
F J
FJ
Donnelly
M
M
Schiffer
M
M
Anderson
L E
LE
D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008027:Light; D008291:Malate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010788:Photosynthesis; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D004220:Disulfides; D011994:Recombinant Proteins; D003958:Diamide; D008291:Malate Dehydrogenase; D004229:Dithiothreitol