title: Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II.
abstract: Prenyltransferases that catalyze the fundamental chain elongation reaction in the isoprenoid biosynthetic pathway contain several highly conserved amino acids, including two aspartate-rich regions thought to be involved in substrate binding and catalysis. We report a study of site-directed mutants for yeast farnesyl-diphosphate synthase (FPPSase; geranyl-diphosphate:isopentenyl-diphosphate, EC 2.5.1.10), a prenyltransferase that catalyzes the sequential 1'-4 coupling of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate and geranyl diphosphate. A recombinant form of FPPSase extended by a C-terminal -Glu-Glu-Phe alpha-tubulin epitope (EEF in single-letter amino acid code) was engineered to facilitate rapid purification of the enzyme by immunoaffinity chromatography and to remove traces of contaminating activity from wild-type FPPSase in the Escherichia coli host. Ten site-directed mutants were constructed in FPPSase::EEF. The six aspartates in domain I (at positions 100, 101, and 104) and domain II (at positions 240, 241, and 244) were changed to alanine (mutants designated D100A, D101A, D104A, D240A, D241A, and D244A); three arginine residues were changed, Arg-109 and Arg-110 to glutamine and Arg-350 to alanine (mutants designated R109Q, R110Q, and R350A); and Lys-254 was converted to alanine (mutant designated K254A). Mutations of the aspartatic residues and nearby arginine residues in domain I and Asp-240 and Asp-241 in domain II drastically lowered the catalytic activity of FPPSase::EEF. The D244A and K254A mutants were substantially less active, while kcat and the Michaelis constants for the R350A mutant were similar to those of FPPSase::EEF. Addition of an -EEF epitope to the C terminus of wild-type FPPSase resulted in a 14-fold increase of KmIPP and a 12-fold decrease of kcat, suggesting that the conserved hydrophilic C terminus of the enzyme may have a role in substrate binding and catalysis.
authors.affiliation: Department of Chemistry, University of Utah, Salt Lake City 84112.
authors.forename: L
authors.initials: L
authors.lastname: Song
authors.forename: C D
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D051228:Geranyltranstransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase