Site-directed mutagenesis reveals the involvement of an additional thioredoxin-dependent regulatory site in the activation of recombinant sorghum leaf NADP-malate dehydrogenase.
The chloroplastic enzyme NADP-malate dehydrogenase is activated by a reversible thiol/disulfide interchange with reduced thioredoxin. Its target disulfide bridge is considered to be located at the amino terminus. To further substantiate the regulatory role of this disulfide, site-directed mutagenesis has been used to replace each or both of the amino-terminal cysteines of the sorghum leaf NADP-malate dehydrogenase, expressed in Escherichia coli, by serines. A truncation mutant lacking the amino terminus has also been produced. Surprisingly, the mutant proteins still required activation by reduced thioredoxin. However, their activation was almost instantaneous, whereas the native enzyme reached full activity after a 10-20 min preincubation. The 8 1/2 for reduced thioredoxin was decreased 2-fold in the mutants, but their Km values for NADPH and oxaloacetate did not change significantly. The inhibition of activation by NADP and inhibition of activity by thiol-derivatizing agents were also retained. These results are interpreted as an indication that two thioredoxin-dependent reduction steps are involved in NADP-dependent malate dehydrogenase light activation, hence that two disulfides per monomer participate in the process. The overall activation rate would depend on a conformational change following the reduction of the amino-terminal disulfide bridge. The amino terminus also plays a role in the dimerization of the protein.
Issakidis
E
E
Laboratoire de Physiologie Végétale Moléculaire, Université de Paris-Sud, Orsay, France.
Miginiac-Maslow
M
M
Decottignies
P
P
Jacquot
J P
JP
Crétin
C
C
Gadal
P
P
D001483:Base Sequence; D004247:DNA; D004220:Disulfides / Q000378:metabolism; D002523:Edible Grain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000528:radiation effects; D008027:Light; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D013879:Thioredoxins; D004247:DNA; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)