title: Significance of Phe-220 and Gln-221 in the catalytic mechanism of farnesyl diphosphate synthase of Bacillus stearothermophilus.
abstract: Farnesyl diphosphate synthase [EC 2.5.1.10] from Bacillus stearothermophilus was specifically altered at two amino acid residues by using site-directed mutagenesis. The highly conserved Phe and Gln residues at the sequential amino acid positions 220-221 in an upstream part of the putative substrate binding site were replaced with Ala and Glu, respectively. These mutageneses (F220A and Q221E) resulted in 10(-5) and 10(-3) decreases in catalytic activity of farnesyl diphosphate synthesis, respectively. Michaelis constants of the Q221E mutant for the allylic substrates (dimethylallyl- and geranyl diphosphates) increased approximately 25- and 2-folds, respectively, compared to wild type, whereas those for the homoallylic substrate (isopentenyl diphosphate) were not altered much. These results suggest that the Phe-Gln motif is involved not only in the binding of allylic substrates but also in the catalysis by farnesyl diphosphate synthase.
authors.affiliation: Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, Sendai, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Koyama
authors.forename: M
authors.initials: M
authors.lastname: Tajima
authors.forename: T
authors.initials: T
authors.lastname: Nishino
authors.forename: K
authors.initials: K
authors.lastname: Ogura
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D051228:Geranyltranstransferase; D005973:Glutamine; D045782:Hemiterpenes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009943:Organophosphorus Compounds; D010649:Phenylalanine; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D013329:Structure-Activity Relationship; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D005973:Glutamine; C043060:3,3-dimethylallyl pyrophosphate; D010649:Phenylalanine; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase