Selection of stabilized 3-isopropylmalate dehydrogenase of Saccharomyces cerevisiae using the host-vector system of an extreme thermophile, Thermus thermophilus.
A leuB strain of Thermus thermophilus TTY1, was transformed with a plasmid vector that directed expression of 3-isopropylmalate dehydrogenase (IPMDH) of Saccharomyces cerevisiae encoded by the LEU2 gene. The original strain could not grow at 50 degrees C without leucine, probably because of the low stability of S. cerevisiae IPMDH. The mutants that could grow without leucine were selected at 50 degrees, 60 degrees, 62 degrees, 65 degrees, 67 degrees, and 70 degrees C, step by step. All the mutant strains except for one isolated at 50 degrees C accumulated mutations. Mutations were serially accumulated: Glu255Val, Asn43Tyr, Ala62Thr, Asn110Lys, and Alal 12Val, respectively, at each step. The analyses of residual activity after heat treatment and the denaturation profile as monitored by circular dichroism showed that thermal stability was increased with accumulation of the mutations. The kinetic parameters of most mutant enzymes were similar to those of the wild type. However, some mutant enzymes showed a reverse correlation between stability and activity: the enzymes with a large increase in thermal stability showed lower activity. Although the wild-type enzyme is unstable in the absence of glycerol, the stabilizing effect of glycerol was not observed for all the mutant enzymes containing the Glu255Val substitution, which is assumed to be located at the hydrophobic interface between two subunits.
Tamakoshi
M
M
Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Hachioji, Japan.
Nakano
Y
Y
Kakizawa
S
S
Yamagishi
A
A
Oshima
T
T
D003001:Cloning, Molecular; D004795:Enzyme Stability; D005800:Genes, Fungal; D005822:Genetic Vectors; D005990:Glycerol; D006358:Hot Temperature; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D009154:Mutation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D016963:Thermus thermophilus / Q000235:genetics*; D014170:Transformation, Genetic
D006836:Hydro-Lyases; C021455:3-isopropylmalate dehydratase; D007930:Leucine; D005990:Glycerol