Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.
Cinnamomum camphora/enzymology* ; Alkyl and Aryl Transferases/chemistry*

Genome mining for the search and discovery of two new globin-like enzymes, TriB fromFusarium poae and TutaA from Schizophyllum commne, are involved in the synthesis of two linear terpenes tricinonoic acid (1) and 2-butenedioic acid (3). Both in vivo heterologous biosynthesis and in vitro biochemical assays showed that these two enzymes catalyzed the C-C double bond cleavage of a cyclic sesquiterpene precursor (-)-germacrene D (7) and a linear diterpene backbone schizostain (2), respectively. Our work presents an unusual formation mechanism of linear terpenes from fungi and expands the functional skills of globin-like enzymes in the synthesis of terpene compounds.
Terpenes/chemistry* ; Alkyl and Aryl Transferases ; Globins ; Diterpenes

To construct an engineered Saccharomyces cerevisiae producing high titres of amorpha-4,11-diene, we investigated the possible synergistic effect of different vectors containing amorpha-4,11-diene synthase(ADS) gene within one yeast cell. We constructed the ADS recombinant plasmid pGADADS. This plasmid and another ADS recombinant plasmid pYeDP60/G/ADS were alone, or co-transformed into yeast Saccharomyces cerevisiae W303-1B and WK1, respectively, resulting in the following engineered yeasts, W303B[pGADADS], W303B[pYGADS], W303B[pYGADS+pGADADS], WK1[pGADADS], WK1[pYGADS] and WK1[pYGADS+pGADADS]. All of the six strains were cultured for GC-MS analysis of amorpha-4,11-diene. The results showed that all of the engineered yeasts could produce amorpha-4,11-diene. The yield of the product was improved with increasing ADS gene copies while no deleterious effect on the strain growth was found. Moreover, the product yield of the engineered yeast co-transformed with multiple plasmids was much higher than the total yield of the different engineered yeasts with only one plasmid, respectively. In conclusion, there was a distinct synergistic effect between different recombinant ADS plasmids within one cell. Our results facilitate the construction of the engineered yeast with high yield of amorpha-4,11-diene, the precursor of artemisinin.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Artemisinins ; chemistry ; metabolism ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism

AIMDesign, synthesis and evaluation of a series of 7-imidazolylalkanamido-1-carboxylalkylbenzodiazepine farnesyltransferase (FTase) inhibitors.

METHODS AND RESULTSCoupling of imidazolylalkylcarboxylic acids and 1-substituted 7-aminobenzodiazepines (5a-5c) yielded 10 new compounds (6-12, 16-18) which were biologically tested against FTase using scintillation proximity assay method.

CONCLUSIONFive target compounds were found to be potential farnesyltransferase inhibitors.

Alkyl and Aryl Transferases ; antagonists & inhibitors ; drug effects ; Benzodiazepines ; chemical synthesis ; chemistry ; pharmacology ; Farnesyltranstransferase ; Imidazoles ; chemical synthesis ; chemistry ; pharmacology ; Inhibitory Concentration 50 ; Molecular Conformation ; Molecular Structure ; Structure-Activity Relationship

AIMTo build a three dimensional structure model that correlates the biological activities and the structures of a series of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta-[1,2-]pyridin-11-yl) piperazines farnesyl protein transferase (FPTase) inhibitors.

METHODS AND RESULTSMutation in the ras oncogene takes place in many human cancers, involving 30%-50% of colon and 90% of pancreatic cancer. Ras proteins function as central switches for signals given by growth factors that direct cell growth and cell differentiation. The dependence of the transforming activity of Ras on the farnesylation has led to intense search for FPTase inhibitors that may have therapeutic pontetial as anticancer agents. This paper is to build a three dimensional structural model that correlates the biological activities and the structures of a series of FPTase inhibitors. The investigated sixty-nine inhibitors contain six types of structures, the optimal conformations of which were studied using system search. A three dimensional quantitative structure-activity relationship (3D-QSAR) model was constructed using the method of comparative molecular field analysis (CoMFA). The resulting cross-validation R2 is 0.581, non-cross-validation R2 0.968, SE 0.148, F 198.7. The predicted activities of 10 inhibitors using this 3D-QSAR model are comparable to the experimental activities, indicating that the 3D-QSAR model has ability to predict activities of new inhibitors and offers an approach to design new FPTase inhibitors.

CONCLUSIONThe information of CoMFA model offers an approach to designing new FPTase inhibitors.

Alkyl and Aryl Transferases ; antagonists & inhibitors ; Enzyme Inhibitors ; chemistry ; pharmacology ; Humans ; Molecular Conformation ; Molecular Structure ; Pyridines ; chemistry ; pharmacology ; Quantitative Structure-Activity Relationship

Sclareol is a member of labdane type diterpenes mostly used as fragrance ingredient. To enable microbial production of sclareol, synthetic pathways were constructed by incorporating labdenediol diphosphate synthase (LPPS) and terpene synthase (TPS) of the plant Salvia sclarea into Saccharomyces cerevisiae. It was found that sclareol production could be benefited by overexpression of key enzyme for precursor biosynthesis, construction of fusion protein for substrate channeling, and removal of signal peptides from LPPS and TPS. Under optimal shake flask culture conditions, strain S6 produced 8.96 mg/L sclareol. These results provided useful information for development of heterologous hosts for production of terpenoids.
Alkyl and Aryl Transferases ; biosynthesis ; genetics ; Diterpenes ; metabolism ; Metabolic Engineering ; methods ; Metabolic Networks and Pathways ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Salvia ; chemistry ; enzymology ; genetics

The expression plasmid pET32CPS harboring SmCPS gene was transformed into E. coli BL21 trxB (DE3) resulting in recombinant strain E. coli [pET32CPS]. The induction of E. coli [pET32CPS] in different temperatures, induction time, IPTG concentrations and A600 values of E. coli were performed. The optimal expression conditions of SmCPS were characterized according to the orthogonal analysis, and the ratio of the interest protein to total proteins reached to 35.6%. The recombinant SmCPS protein purified by Ni2+ affinity chromatography column was identified by SDS-PAGE and Western blotting, and then used for rabbit immunization. The titer of the rabbit antiserum against SmCPS was about 1:24 300 after the third immunization, and could specifically recognize the antigen of SmCPS protein by Western blotting analysis. The successful preparation of polyclonal antibody against SmCPS laid a foundation for further correlative study between expression of SmCPS and the production of tanshinones in protein level.
Alkyl and Aryl Transferases ; genetics ; isolation & purification ; metabolism ; Animals ; Antibody Formation ; Escherichia coli ; metabolism ; Gene Expression ; Immune Sera ; biosynthesis ; immunology ; Isopropyl Thiogalactoside ; chemistry ; Male ; Plant Proteins ; genetics ; isolation & purification ; metabolism ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Plasmids ; Rabbits ; Recombinant Proteins ; genetics ; metabolism ; Salvia miltiorrhiza ; chemistry ; Temperature ; Time Factors ; Transformation, Genetic

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.
1-Phosphatidylinositol 3-Kinase/metabolism ; Alkyl and Aryl Transferases/metabolism ; Animals ; Anticholesteremic Agents/pharmacology ; Cells, Cultured ; Enzyme Inhibitors/metabolism/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Gene Expression Regulation, Enzymologic/*drug effects ; I-kappa B Kinase/antagonists & inhibitors/metabolism ; Macrophages, Alveolar/cytology/*drug effects/*enzymology ; Matrix Metalloproteinase 9/genetics/*metabolism ; Mitogen-Activated Protein Kinase Kinases/metabolism ; Polyisoprenyl Phosphates/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Sesquiterpenes/metabolism ; Signal Transduction/physiology ; Simvastatin/*pharmacology ; Smoke/*adverse effects ; *Tobacco/adverse effects/chemistry