The biosynthesis of rubber: Incorporation of isopentenyl pyrophosphate into purified rubber particles by a soluble latex serum enzyme.
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ABSTRACT:
SUBMITTER: McMullen AI
PROVIDER: S-EPMC1270063 | biostudies-other | 1966 Oct
REPOSITORIES: biostudies-other
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Project description:The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.
| S-EPMC3295384 | biostudies-literature
Project description:Natural rubber is usually synthesized in the rubber particles present in the latex of rubber-producing plants such as the Pará rubber tree (Hevea brasiliensis) and rubber dandelion (Taraxacum kok-saghyz). Since the detailed lipid compositions of fresh latex and rubber particles of the plants are poorly known, the present study reports detailed compound lipid composition, focusing on phospholipids and galactolipids in the latex and rubber particles of the plants. In the fresh latex and rubber particles of both plants, phospholipids were much more dominant (85-99%) compared to galactolipids. Among the nine classes of phospholipids, phosphatidylcholines (PCs) were most abundant, at ~80%, in both plants. Among PCs, PC (36:4) and PC (34:2) were most abundant in the rubber tree and rubber dandelion, respectively. Two classes of galactolipids, monogalactosyl diacylglycerol and digalactosyl diacylglycerol, were detected as 12% and 1%, respectively, of total compound lipids in rubber tree, whereas their percentages in the rubber dandelion were negligible (< 1%). Overall, the compound lipid composition differed only slightly between the fresh latex and the rubber particles of both rubber plants. These results provide fundamental data on the lipid composition of rubber particles in two rubber-producing plants, which can serve as a basis for artificial rubber particle production in the future.
| S-EPMC7662343 | biostudies-literature
Project description:1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K(m) 0.042mm), 2.0mm-ATP (K(m) 0.19mm) and 8mm-Mg(2+) at 40 degrees C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5-6.5 with 0.1mm-5-pyrophosphomevalonate (K(m) 0.004mm), 1.5mm-ATP (K(m) 0.12mm) and 2mm-Mg(2+). The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.
| S-EPMC1177155 | biostudies-other
Project description:Isopentenyl pyrophosphate isomerase (EC 5.3.3.2) was purified from extracts of pig liver by ammonium sulphate fractionation and by gel filtration. After about 20-fold purification the preparations were free of phosphatase and prenyltransferase (EC 2.5.1.1), the two enzymes that could have interfered with the assays. The isomerase has a distinct pH optimum at 6.0 and is activated by Mn(2+) in preference to Mg(2+). The K(m) value for isopentenyl pyrophosphate is 4x10(-6)m. The equilibrium of the reaction favours the formation of dimethylallyl pyrophosphate. The reversibility of the isomerase reaction was demonstrated directly by the formation of isopentenyl pyrophosphate from dimethylallyl pyrophosphate. It is suggested that two prenyl isomerases might exist, one involved in the synthesis of trans- and another in the synthesis of cis-polyprenyl substances.
| S-EPMC1198587 | biostudies-other
Project description:1. Isopentenol was converted into isopentenyl phosphate with phosphoryl chloride in ether containing pyridine. 2. The isopentenyl phosphate reacted in 2-methylpropan-2-ol-water with morpholine and dicyclohexylcarbodi-imide to give isopentenyl phosphoromorpholidate. 3. The isopentenyl phosphoromorpholidate, with inorganic phosphate in pyridine containing tributylamine, gave isopentenyl pyrophosphate. The yield of pyrophosphate from monophosphate was 80-85% and the yield of pyrophosphate from isopentenol 40-60%. [1-(14)C]-Isopentenyl pyrophosphate was prepared by this method. 4. The yield of isopentenyl pyrophosphate from isopentenyl phosphate was substantially improved, in comparison with the yields obtained by published methods via the phosphoramidate, by the use of the phosphoromorpholidate.
| S-EPMC1198343 | biostudies-other
Project description:1. beta-Hydroxy-beta-methyl[3-(14)C]glutaryl-CoA is efficiently incorporated into rubber on incubation with Hevea brasiliensis latex, and the incorporation is diminished in the presence of unlabelled mevalonate. beta-Hydroxy-beta-methylglutaric acid is not utilized for rubber synthesis, but inhibits the formation of rubber from beta-hydroxy-beta-methylglutaryl-CoA. 2. The incorporation of beta-hydroxy-beta-methylglutaryl-CoA into rubber is stimulated equally by NADP(+) and NADPH and less so by NAD(+) and NADH. ATP is slightly stimulatory and CoA is inhibitory. 3. beta-Hydroxy-beta-methylglutaryl-CoA reductase is concentrated in the sediment (bottom fraction) formed by centrifuging latex at low speed and the enzyme is unstable in the absence of cysteine or GSH. The formation of NADPH takes place in the latex serum. 4. There is a marked seasonal variation in the extent of beta-hydroxy-beta-methylglutaryl-CoA incorporation into rubber in latex, but mevalonate incorporation is relatively constant. This observation, together with the finding that beta-hydroxy-beta-methylglutaryl-CoA reduction is the rate-limiting step in the formation of rubber from beta-hydroxy-beta-methylglutaryl-CoA, suggests that the conversion of beta-hydroxy-beta-methylglutaryl-CoA into mevalonate is of importance in the regulation of rubber synthesis. 5. Evidence suggesting that beta-hydroxy-beta-methylglutaryl-CoA lyase is present in H. brasiliensis latex has been obtained.
| S-EPMC1184865 | biostudies-other
Project description:Isopentenyl pyrophosphate (IPP) is the universal C5 precursor for isoprenoids, the largest class of natural products and frequent targets for metabolic engineering. In engineered microbial systems, IPP toxicity presents a challenge since its formation is unavoidable in the production of high-value, long-chain terpenes. In this work, we develop an experimental platform to study IPP toxicity in E. coli engineered to produce isoprenol, an IPP-derived C5 alcohol. We first characterize the physiological response to IPP accumulation, demonstrating that elevated levels of IPP are linked to growth inhibition, altered morphology, and reduced cell viability. We show that IPP toxicity selects for pathway “breakage”, using proteomics to identify a reduction in phosphomevalonate kinase (PMK) as a probable recovery mechanism. Next, we demonstrate that endogenous E. coli metabolism is globally impacted by IPP accumulation, which results in inhibited nutrient uptake, reduced ATP levels, and an apparent “pause” in metabolism. Finally, we suggest that IPP toxicity is mediated by the formation of an isoprenyl-ATP analog (ApppI). The complementary data presented here represent the most comprehensive assessment of IPP stress to date and suggest potential strategies for the alleviation of IPP and prenyl diphosphate toxicity.
2018-03-14 | GSE102672 | GEO
Project description:Biodegradation of rubber (polyisoprene) is initiated by oxidative cleavage of the polyisoprene backbone and is performed either by an extracellular rubber oxygenase (RoxA) from Gram-negative rubber degrading bacteria or by a latex clearing protein (Lcp) secreted by Gram-positive rubber degrading bacteria. Only little is known on the biochemistry of polyisoprene cleavage by Lcp and on the types and functions of the involved cofactors.A rubber-degrading bacterium was isolated from the effluent of a rubber-processing factory and was taxonomically identified as a Rhodococcus rhodochrous species. A gene of R. rhodochrous RPK1 that coded for a polyisoprene-cleaving latex clearing protein (lcp Rr ) was identified, cloned, expressed in Escherichia coli and purified. Purified LcpRr had a specific activity of 3.1 U/mg at 30 °C and degraded poly(1,4-cis-isoprene) to a mixture of oligoisoprene molecules with terminal keto and aldehyde groups. The pH optimum of LcpRr was higher (pH 8) than for other rubber-cleaving enzymes (? pH 7). UVvis spectroscopic analysis of LcpRr revealed a cytochrome-specific absorption spectrum with an additional feature at long wavelengths that has not been observed for any other rubber-cleaving enzyme. The presence of one b-type haem in LcpRr as a co-factor was confirmed by (i) metal analysis, (ii) solvent extraction, (iii) bipyridyl assay and (iv) detection of haem-b specific m/z values via mass-spectrometry.Our data point to substantial differences in the active sites of Lcp proteins obtained from different rubber degrading bacteria.
| S-EPMC4877957 | biostudies-literature
Project description:Staphylococcus aureus virulence has been associated with the production of phenol soluble modulins (PSM). PSM are known to activate, attract and lyse neutrophils. However, the functional characterizations were generally performed in the absence of human serum. Here, we demonstrate that human serum can inhibit all the previously-described activities of PSM. We observed that serum can fully block both the cell lysis and FPR2 activation of neutrophils. We show a direct interaction between PSM and serum lipoproteins in human serum and whole blood. Subsequent analysis using purified high, low, and very low density lipoproteins (HDL, LDL, and VLDL) revealed that they indeed neutralize PSM. The lipoprotein HDL showed highest binding and antagonizing capacity for PSM. Furthermore, we show potential intracellular production of PSM by S. aureus upon phagocytosis by neutrophils, which opens a new area for exploration of the intracellular lytic capacity of PSM. Collectively, our data show that in a serum environment the function of PSM as important extracellular toxins should be reconsidered.
| S-EPMC3310821 | biostudies-literature
Project description:Two types of enzyme for oxidative cleavage of poly(cis-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by Xanthomonas sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome c peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833-13838, 2013, http://dx.doi.org/10.1073/pnas.1305560110). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of Streptomyces sp. strain K30 in a ?roxA background of Xanthomonas sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C20 and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack.
| S-EPMC4135767 | biostudies-literature