Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis.

Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules.

Cloning and characterization of a farnesyl pyrophosphate synthase from Matricaria recutita L. and its upregulation by methyl jasmonate.

Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.

Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

Farnesyl diphosphate synthase assay.

Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts.

Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel.

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.

[Molecular cloning of farnesyl pyrophosphate synthase from Alisma orientale (Sam.) Juzep. and its distribution pattern and bioinformatics analysis].

Triterpenes, which have large application potential in the treatment of cancer, are the main active components of genuine medicinal material Alisma orientale (Sam.) Juzep. Farnesyl pyrophosphate synthase (FPPS) is one of the important rate-limiting enzymes in the synthetic pathway of triterpenes. In this study the FPPS full length cDNA of the A. orientale, was cloned via homology-based cloning approach and rapid amplification of cDNA ends (RACE). The full length of the FPPS cDNA was 1 531 bp (accession no. HQ724508), which contained a full 1 032 bp ORF that encoded 343 amino acids. The deduced protein sequence exhibited five conserved motifs, two of which is riched of Asp (DDXXD). The result of real-time quantitative PCR (QRT-PCR) showed that FPPS gene was expressed in different organs of A. orientale. The expression increased from October to the first ten-day period of December, and then decreased. The FPPS gene expression was higher in leaves but lower in leafstalk, tuber and root. HPLC analysis of active components 23-acetyl-alismol B of A. orientale. during different periods indicated that its change trend should be consistent with FPPS gene expression. It can be primarily deduced that FPPS gene should be an important control point in the synthetic pathway of Alisma terpenes. This study may facilitate the quality of medicinal plants through gene engineering in the future.

Molecular cloning, characterization, and differential expression of a farnesyl-diphosphate synthase gene from the basidiomycetous fungus Ganoderma lucidum.

A farnesyl-diphosphate synthase gene, designated GlFPS, was isolated from a triterpene-producing basidiomycetous fungus, Ganoderma lucidum. The GlFPS cDNA was found to contain an open reading frame of 1,083 bp, encoding a protein of 360 amino acids with a calculated molecular mass of 41.27 kDa. The deduced amino acid sequence of the GlFPS cDNA exhibited a high homology with other fungal FPS genes, and contained four conserved domains. Phylogenetic analysis showed that GlFPS belonged to the basidiomycete FPS group. Competitive PCR revealed that GlFPS was constitutively expressed in the mycelium growth stage, whereas the transcripts of GlFPS accumulated to high levels rapidly during the process of mushroom primordia. Treatment of mycelia with exogenous methyl jasmonate also caused a large accumulation of GlFPS mRNA. Subsequently, promoter analysis indicated that the 5' upstream region of GlFPS possessed various potential regulatory elements associated with physiological and environmental factors. Functional complementation of GlFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl-diphosphate synthase.

Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases.

Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [(14)C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.