dolichols and isopentenyl-pyrophosphate

Dolichol and natural rubber are representative cis-polyisoprenoids in primary and secondary metabolism, respectively. Their biosynthesis is catalyzed by cis-prenyltransferase (CPT) by sequential condensations of isopentenyl diphosphates (IPPs) to a priming molecule. Although prokaryotic CPTs have been well characterized, the mechanism of eukaryotic CPTs in cis-polyisoprene biosynthesis was only recently revealed. It was shown that eukaryotes have evolved a unique protein complex, comprised of CPT and CPT-binding protein (CBP), to synthesize cis-polyisoprenoids. In the context of this new discovery, we found discrepancies in literature for CPT or CBP biochemical assays and in vivo CPT complementation using rer2 (yeast CPT) yeast mutant. Our study here shows that rer2 revertants occur at a frequency that cannot be disregarded and are likely accountable for the results that cannot be explained by the CPT/CBP heteroprotein complex model. To make a stable mutant, SRT1 gene (secondary CPT expressed at a basal level in yeast) was additionally deleted in the rer2Δ mutant background. This stable rer2Δ srt1Δ strain was then used to individually or simultaneously express Arabidopsis CPT1 (AtCPT1, At2g17570) and CBP (AtLEW1, At1G11755). We found that the simultaneous expression of Arabidopsis CPT1 and AtLEW1 effectively complements the rer2Δ srt1Δ strain, whereas the individual expression of AtCPT1 alone or AtLEW1 alone failed to rescue the yeast mutant. Microsomes from the dual expresser showed an efficient incorporation of IPPs into cis-polyisoprenoid (30% in 2h). These results showed that the CPT/CBP heteroprotein complex model is valid in Arabidopsis thaliana. Experimental details of these results are described in this methodology paper.

Topics: Alkyl and Aryl Transferases; Arabidopsis; Arabidopsis Proteins; Biosynthetic Pathways; Dimethylallyltranstransferase; Dolichols; Gene Knockdown Techniques; Hemiterpenes; Mutation; Organophosphorus Compounds; Rubber; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Secondary Metabolism; Transferases

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C-isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.

Topics: Carbon Radioisotopes; Chromatography, Thin Layer; Detergents; Dolichols; Hemiterpenes; Kinetics; NADP; Organophosphorus Compounds; Radioisotope Dilution Technique; Saccharomyces cerevisiae; Tritium

The alpha-saturation reaction involved in the biosynthesis of dolichol has been investigated with rat liver preparations. Under improved in vitro conditions with 10,000 x g supernatant of rat liver homogenates in the presence of NADPH at pH 8.0, dolichol was synthesized from isopentenyl diphosphate and Z,E,E-geranylgeranyl diphosphate. Neither dolichyl diphosphate nor dolichyl phosphate was detected. The chain length distribution of the dolicohol was the same as that of dehydrodolichyl products. In an assay system containing dehydrodolichol, dehydrodolichyl phosphate, or dehydrodolichyl diphosphate as a substrate, dehydrodolichol was predominantly converted into dolichol. The enzyme that catalyzes the conversion of dehydrodolichol to dolichol was localized in microsomes. The reductase activity was stimulated 9-fold by the addition of a 100,000 x g soluble fraction. The reductase had an opimal pH at 8.0. These results indicate that dolichol is formed from dehydrodolichol in rat liver microsomes. The formation of dolichol from dehydrodolichol was also catalyzed by 10,000 x g supernatant of rat or pig testis homogenates.

Topics: Animals; Chromatography, Ion Exchange; Dolichols; Hemiterpenes; Kinetics; Liver; Male; Microsomes, Liver; NADP; Organ Specificity; Organophosphorus Compounds; Oxidoreductases; Polyisoprenyl Phosphates; Rats; Rats, Sprague-Dawley; Subcellular Fractions; Substrate Specificity; Swine; Testis

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.

Topics: Animals; Cations, Divalent; Detergents; Dimethylallyltranstransferase; Dolichols; Hemiterpenes; Intracellular Membranes; Kinetics; Male; Microsomes, Liver; Octoxynol; Organophosphorus Compounds; Polyethylene Glycols; Polyisoprenyl Phosphates; Rats; Rats, Inbred Strains; Sesquiterpenes