RESUMO
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).
Assuntos
Agaricales/metabolismo , Alquil e Aril Transferases/biossíntese , Borracha/metabolismo , Agaricales/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/isolamento & purificação , Sequência de Aminoácidos , Autorradiografia , Southern Blotting , Cromatografia em Camada Fina , DNA Complementar/biossíntese , DNA Complementar/genética , Dimetilaliltranstransferase/biossíntese , Dimetilaliltranstransferase/genética , Eletroforese em Gel de Poliacrilamida , Geraniltranstransferase , Cinética , Dados de Sequência Molecular , Peso Molecular , RNA/biossíntese , RNA/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A structural study of low molecular weight rubbers from Jackfruit (Artocarpus heterophyllus) and Painted spurge (Euphorbia heterophylla) was carried out as model compounds of natural rubber from Hevea brasiliensis. The rubber content of latex from Jackfruit was 0.4-0.7%, which is very low compared with that of 30-35% in the latex from Hevea tree. The rubber from Jackfruit latex was low molecular weight with narrow unimodal molecular weight distribution (MWD), whereas that obtained from E. heterophylla showed very broad MWD. The 1H and 13C NMR analyses showed that Jackfruit rubber consists of a dimethylallyl group and two trans-isoprene units connected to a long sequence of cis-isoprene units. The alpha-terminal group of Jackfruit rubber was presumed to be composed of a phosphate group based on the presence of 1H NMR signal at 4.08 ppm corresponding to the terminal =CH-CH2OP group.
