A thioredoxin-independent fully active NADP-malate dehydrogenase obtained by site-directed mutagenesis.
A triple cysteine mutant of sorghum leaf NADP-malate dehydrogenase has been constructed by site-directed mutagenesis, combining the previously obtained mutation of the two N-terminal cysteines with the mutation of the most internal of the two C-terminal cysteines. The construct, over-expressed in E. coli, yielded an always active, dithiol-insensitive enzyme. It can be concluded that the dithiol activation of the unmodified enzyme involves a maximum of two different disulfides per subunit, and that none of the mutated cysteines is implicated in catalysis.
Issakidis
E
E
Laboratoire de Physiologie Végétale Moléculaire, URA 1128 CNRS, Université de Paris-Sud, Orsay, France.
Decottignies
P
P
Miginiac-Maslow
M
M
D001483:Base Sequence; D003545:Cysteine / Q000737:chemistry; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D010944:Plants / Q000201:enzymology; D013329:Structure-Activity Relationship; D013879:Thioredoxins / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010940:Plant Proteins; D013879:Thioredoxins; D009249:NADP; D008291:Malate Dehydrogenase; D003545:Cysteine