Cloning and characterization of farnesyl diphosphate synthase from the rubber-producing mushroom Lactarius chrysorrheus.
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).
Mekkriengkrai
Dararat
D
Department of Chemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
Sando
Tomoki
T
Hirooka
Kazutake
K
Sakdapipanich
Jitladda
J
Tanaka
Yasuyuki
Y
Fukusaki
Ei-Ichiro
E
Kobayashi
Akio
A
D000363:Agaricales / Q000737:chemistry / Q000378:metabolism*; D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001345:Autoradiography; D015139:Blotting, Southern; D002855:Chromatography, Thin Layer; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004122:Dimethylallyltranstransferase / Q000096:biosynthesis / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D051228:Geranyltranstransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012408:Rubber / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D012313:RNA; D012408:Rubber; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; D051228:Geranyltranstransferase