title: Identification of significant residues in the substrate binding site of Bacillus stearothermophilus farnesyl diphosphate synthase.
abstract: Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al. (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R). The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively. Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold. Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type. These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function. Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction.
authors: Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, Sendai, Japan.
authors: T
authors: T
authors: Koyama
authors: M
authors: M
authors: Tajima
authors: H
authors: H
authors: Sano
authors: T
authors: T
authors: Doi
authors: A
authors: A
authors: Koike-Takeshita
authors: S
authors: S
authors: Obata
authors: T
authors: T
authors: Nishino
authors: K
authors: K
authors: Ogura
mesh_terms: D000080462:Acyclic Monoterpenes; D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002855:Chromatography, Thin Layer; D017124:Conserved Sequence; D004224:Diterpenes / Q000378:metabolism; D005204:Farnesol / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D051228:Geranyltranstransferase; D045782:Hemiterpenes; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds / Q000378:metabolism; D016415:Sequence Alignment; D013729:Terpenes / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D000080462:Acyclic Monoterpenes; D004224:Diterpenes; D045782:Hemiterpenes; D007211:Indoles; D009943:Organophosphorus Compounds; D013729:Terpenes; C043060:3,3-dimethylallyl pyrophosphate; C041272:indolepropanol phosphate; D005204:Farnesol; C017338:geranylgeraniol; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; C007836:geraniol