Expression, purification, and substrate specificity of isocitrate dehydrogenase from Thermus thermophilus HB8.
Isocitrate dehydrogenase (ICDH) from an extreme thermophile, Thermus thermophilus HB8, was overexpressed in Escherichia coli. The enzyme was easily purified to homogeneity by a combination of heat treatment (70 degrees C, 20 min) and column chromatography. The N-terminal sequence of the protein thus purified coincided with that of the protein extracted from the thermophile. The substrate specificity of the enzyme was mutationally analyzed and engineered to recognize 3-alkyl-malate as a substrate. Based on the three-dimensional structure of E. coli isocitrate dehydrogenase, Ser97 qnd Asn99 of the thermophile enzyme were speculated to participate in the substrate recognition, and these residues were replaced with threonine and leucine, respectively. Molecular recognition of the mutant enzymes, [S97T]ICDH, [N99L]ICDH, and [S97T, N99L]ICDH, were studied using isocitrate, 3-isopropylmalate, and 3-ethylmalate. The affinity toward isocitrate was reduced in the cases of [S97T]ICDH and [N99L]ICDH, confirming the importance of the residues for the reaction. Though none of the mutants acted on 3-isopropylmalate, [N99L]ICDH was competitively inhibited by 3-isopropylmalate with a higher affinity than that of the wild-type enzyme. [N99L]ICDH showed an approximately 10(3)-fold higher value of (kcat/Km)3-ethylmalate/(kcat/Km)isocitrate than the wild-type enzyme, indicating that the single mutation of Asn99 to leucine switched the substrate specificity of the enzyme away from isocitrate and toward 3-ethylmalate.
Miyazaki
K
K
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Yaoi
T
T
Oshima
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression / Q000235:genetics; D005798:Genes, Bacterial; D007521:Isocitrate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007523:Isocitrates / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics; D013912:Threonine / Q000737:chemistry
D007523:Isocitrates; D008293:Malates; D011994:Recombinant Proteins; D013912:Threonine; C025313:beta-isopropylmalate; C034219:isocitric acid; D007521:Isocitrate Dehydrogenase; D007930:Leucine