title: Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8.
abstract: The co-enzyme specificity of 3-isopropylmalate dehydrogenase from an extreme thermophile, Thermus thermophilus HB8, was changed from NAD to NADP by site-directed muta-genesis. Based on sequence comparison of 3-isopropylmalate dehydrogenases from various organisms with NAD- and NADP-dependent isocitrate dehydrogenases, Ser226, Ser253 and Ile279 of 3-isopropylmalate dehydrogenase were suggested as determining the co-enzyme specificity. These residues were replaced with the corresponding residues of NADP-dependent isocitrate dehydrogenases; Arg, Gly and Tyr respectively. The single-mutated enzymes, S226R and 1279Y, enhanced the activities towards NADP approximately 10- and approximately 3-fold respectively, whereas S253G reduced the activity. Among the multiple-mutated enzymes, the double-mutated S226R/I279Y increased the catalytic efficiency against NADP (approximately 5-fold) and shifted the specificity for NAD towards NADP most significantly (approximately 173-fold).
authors.affiliation: Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000494:pharmacology*; D009249:NADP / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D009243:NAD; D009249:NADP; D000429:Alcohol Oxidoreductases; D007521:Isocitrate Dehydrogenase; D050539:3-Isopropylmalate Dehydrogenase