Novel substrate specificity of designer 3-isopropylmalate dehydrogenase derived from Thermus thermophilus HB8.
Redesigning of an enzyme for a new catalytic reaction and modified substrate specificity was exploited with 3-isopropylmalate dehydrogenase (IPMDH). Point-mutation on Gly-89, which is not in the catalytic site but near it, was done by changing it to Ala, Ser, Val, and Pro, and all the mutations changed the substrate specificity. The mutant enzymes showed higher catalytic efficiency (kcat/Km) than the native IPMDH when malate was used as a substrate instead of 3-isopropylmalate. More interestingly, an additional insertion of Gly between Gly-89 and Leu-90 significantly altered the substrate-specificity, although the overall catalytic activity was decreased. Particularly, this mutant turned out to efficiently accept D-lactic acid, which was not accepted as a substrate by wild-type IPMDH at all. These results demonstrate the opportunity for creating nove,enzymes by modification of amino acid residues that do not directly participate in catalysis, or by insertion of additional residues.
Fujita
M
M
Department of Chemistry, Tokyo Institute of Technology, Japan.
Tamegai
H
H
Eguchi
T
T
Kakinuma
K
K
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology*
D017931:DNA Primers; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase