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A thioredoxin‐independent fully active NADP‐malate dehydrogenase obtained by site‐directed mutagenesis
Author(s) -
Issakidis Emmanuelle,
Decottignies Paulette,
Miginiac-Maslow Myroslawa
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80620-a
Subject(s) - mutagenesis , site directed mutagenesis , cysteine , biochemistry , malate dehydrogenase , mutant , enzyme , thioredoxin , directed mutagenesis , chemistry , mutation , active site , dehydrogenase , biology , gene
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.