Showing papers on "Lanosterol synthase published in 2013"

Purification, kinetics, inhibitors and CD for recombinant β‐amyrin synthase from Euphorbia tirucalli L and functional analysis of the DCTA motif, which is highly conserved among oxidosqualene cyclases

Abstract: β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations.

Zebrafish etv7 regulates red blood cell development through the cholesterol synthesis pathway

Abstract: ETV7 is a human oncoprotein that cooperates with Eμ-MYC to promote pre-B-cell leukemia in mice. It is normally expressed in the bone marrow and fetal liver and is upregulated in primary leukemia, suggesting that it is involved in proper hematopoiesis and leukemogenesis. ETV7 has been deleted in most rodents, but is conserved in all other vertebrates, including the zebrafish, Danio rerio. In this report, we characterize the function of the zebrafish etv7 gene during erythropoiesis. Our results demonstrate that etv7 regulates the expression of the zebrafish lanosterol synthase (lss) gene, an essential gene in the cholesterol synthesis pathway. Furthermore, morpholino knockdown of etv7 leads to loss of hemoglobin-containing red blood cells, a phenotype that can be rescued by injection of exogenous cholesterol. We conclude that etv7 is essential for normal red blood cell development through regulation of the lss gene and the cholesterol synthesis pathway.

Recent Advances in Study of Ginsenoside Biosynthetic Pathway in Panax ginseng

Abstract: Ginsenosides, the major bioactive ingredients of P. ginseng can improve the anti-disease abilities of human being, and generate significant social and economic benefits. However, along with gradually or rapidly or dramatically increasing demand of the ginsenosides, extensive studies have focused on regulating the ginsenoside biosynthetic pathway on a genetic level. This review provides the latest research progress on biosynthetic pathway of ginsenosides, including the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathway, which is newly discovered and located in P. ginseng. Moreover, it also indicated lanosterol synthase metabolic flux present in P. ginseng.

Progress in Understanding of the Key Enzyme Genes of Ginsenoside Biosynthesis in Panax ginseng

Abstract: Ginsenosides, the major bioactive ingredients of P. ginseng can improve the anti-disease abilities of human being, and generate significant social and economic benefits. However, along with gradually or rapidly or dramatically increasing demand of the ginsenosides, extensive studies have focused on regulating the ginsenoside biosynthetic pathway on a genetic level. In this article, ginsenoside biosynthesis of key enzyme genes are described, including squalene synthase (SS), squalene epoxidase (SE), oxidosqualene cyclase (OSC), dammarenediol synthase (DS), β-amyrin synthase (β-AS), lanosterol synthase (LAS), cycloartenol synthase (CAS) and P450. Additionally, this review critically analyzes and evaluates the background and theoretical basis of the previous researches, as well as the deficiencies of these researches.