Conversion from Farnesyl Diphosphate Synthase to Geranylgeranyl Diphosphate Synthase by Random Chemical Mutagenesis

Abstract

Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce prenyl diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.

Maize cDNAs Expressed in Endosperm Encode Functional Farnesyl Diphosphate Synthase with Geranylgeranyl Diphosphate Synthase Activity

“…The conversion of archaeal GGPPS to FPPS by sitedirected mutagenesis identified key residues involved in determining product length specificity, a result that in turn served as the basis for a hypothesis on the evolution of eubacterial and eukaryotic FPPS types from an ancestral GGPPS (Ohnuma et al, 1997). Chemical random mutagenesis was used to determine the conversion of FPPS to GGPPS in Bacillus stearothermophylus (Ohnuma et al, 1996). The mutation responsible for the modified enzymatic activity was found at a position equivalent to crucial residues in other isoprenoid synthases.…”

Crystal Structure of Octaprenyl Pyrophosphate Synthase from Hyperthermophilic Thermotoga maritima and Mechanism of Product Chain Length Determination

“…1). The corresponding residue at this position for FPPs from Bacillus stearothermophilus is a large Tyr, and when changed to smaller residue such as Gly and Ala the product chain length was increased the most (22,23). In addition to the fifth amino acid residue, sixth and eighth positions before the first DDXXD have also been shown important to control the product specificity of archael FPPs and C 20 -GGPPs (24).…”

A Pathway Where Polyprenyl Diphosphate Elongates in Prenyltransferase

“…In both Sulfolobus acidocaldarius GGPP synthase and Bacillus stearothermophilus FPP synthase, we have found that the fifth amino acid before the first aspartate-rich motif (FARM) is extremely important for the ultimate chain length determination (21,22). From the analysis of the amino acid, we demonstrated that the terminus of an elongating allylic product directly contacts with this amino acid residue, and the interaction must prevent further condensation of IPP (23).…”

“…The mutated FPP synthases were prepared according to the previously reported method (8,21,23). The assay mixture contained, in a final volume of 200 l, 25 nmol of [1- , and a suitable amount of enzyme.…”

“…Although we examined various FPP synthases and GGPP synthases mutated at the 5th position before FARM, there was no mutated enzyme that could yield any products longer than C 30 (21)(22)(23). Therefore, we hypothesized that another amino acid upstream from FARM must block the further elongation and that the alteration of this amino acid would be essential to create from GGPP synthase and FPP synthase an enzyme that produces compounds longer than C 30 .…”