Cloning and Characterization of Farnesyl Diphosphate Synthase from the Rubber-Producing MushroomLactarius chrysorrheus

Abstract

Farnesyl diphosphate is involved in rubber biosynthesis as an initiating substrate for both polyprenol and mushroom rubber. So far, we have isolated the cDNA of a farnesyl diphosphate synthase (FPS) for the first time from a rare rubber-producing mushroom, Lactarius chrysorrheus, by the degenerate RT-PCR technique based on sequence information of FPS genes from fungi and yeasts. The open reading frame was clarified to encode a protein of 381 amino acid residues with a calculated molecular weight of 42.9 kDa. The deduced amino acid sequence of L. chrysorrheus FPS showed about 50% identity with those of other fungi and yeasts as well as plants. We expressed the cDNA of L. chrysorrheus FPS in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein. The purified obtained protein showed FPS activity in which geranyl diphosphate (GPP) served as primary substrate, with a 2.4-fold higher k(cat)/K(m) value for GPP than for dimethylallyl diphosphate (DMAPP).

Characterization of Potential Drug Targets Farnesyl Diphosphate Synthase and Geranylgeranyl Diphosphate Synthase in Schistosoma mansoni

“…However, there has not been any reported activity of TbFPPS or TcFPPS with FPP. FPPS from the rubber-producing mushroom Lactarius chrysorrheus has been shown to be unable to form products with FPP as the substrate (48), and data from the Comprehensive Enzyme Information System (http://www.brenda-enzymes.org/) indicate that only DMAPP and GPP are substrates for FPPS enzymes from over 20 different species. However, FPPS enzymes from a number of parasitic protozoa, including Toxoplasma gondii (44), Plasmodium vivax (49), and Cryptosporidium parvum (50), were recently shown to be "bifunctional" prenyl synthases that are capable of accepting DMAPP, GPP, and FPP as substrates to produce FPP and GGPP, as well as, in some cases, products with even longer chains.…”

Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate

“…An aliquot of the extracted hexane phase was then counted using a scintillation counter. In order to analyze the radioactive reaction products, enzymatic reactions were conducted in accordance with the method described by Mekkriengkrai et al (2004). The prenyl diphosphate products were extracted with 1-butanol saturated with water and then treated with potato acid phosphatase at 37 ºC overnight to hydrolyze the diphosphate moiety.…”

Molecular Cloning, Characterization, and Differential Expression of a Farnesyl-Diphosphate Synthase Gene from the Basidiomycetous FungusGanoderma lucidum

“…Relative transcript levels of the G. lucidum FPS gene were determined by competitive PCR. 15,16) Total RNA was extracted from different G. lucidum developmental stages, including primordia and mycelial samples (10,12,14,16,18, and 20 d old), and an additional DNA digestion step was included (RNase Free DNase I, Takara, Dalian, China). Then aliquots of total RNA (1 mg) were reverse-transcribed to cDNA with a PrimeScriptÔ 1st Strand cDNA Synthesis Kit (Takara, Dalian, China).…”

“…FPS genes and cDNAs were first characterized in vertebrates and Saccharomyces cerevisiae, 6) and more recently in many other species, including Arabidopsis thaliana, 7) maize, 8) rice, 9) Lactarius chrysorrheus, 10) dipteran, 11) and Schizosaccharomyces pombe. 12) Comparisons of the amino acid sequences of FPS, ranging from bacteria to higher eukaryotes, have shown that all the FPS known so far contain four distinct regions with high similarity at the amino acid level, two of which are present in all polyprenyl diphosphate synthases.…”