Stilbenoid prenyltransferases define key steps in the diversification of peanut phytoalexins.

Defense responses of peanut (Arachis hypogaea) to biotic and abiotic stresses include the synthesis of prenylated stilbenoids. Members of this compound class show several protective activities in human disease studies, and the list of potential therapeutic targets continues to expand. Despite their medical and biological importance, the biosynthetic pathways of prenylated stilbenoids remain to be elucidated, and the genes encoding stilbenoid-specific prenyltransferases have yet to be identified in any plant species. In this study, we combined targeted transcriptomic and metabolomic analyses to discover prenyltransferase genes in elicitor-treated peanut hairy root cultures. Transcripts encoding five enzymes were identified, and two of these were functionally characterized in a transient expression system consisting of Agrobacterium-infiltrated leaves of Nicotiana benthamiana We observed that one of these prenyltransferases, AhR4DT-1, catalyzes a key reaction in the biosynthesis of prenylated stilbenoids, in which resveratrol is prenylated at its C-4 position to form arachidin-2, whereas another, AhR3'DT-1, added the prenyl group to C-3' of resveratrol. Each of these prenyltransferases was highly specific for stilbenoid substrates, and we confirmed their subcellular location in the plastid by fluorescence microscopy. Structural analysis of the prenylated stilbenoids suggested that these two prenyltransferase activities represent the first committed steps in the biosynthesis of a large number of prenylated stilbenoids and their derivatives in peanut. In summary, we have identified five candidate prenyltransferases in peanut and confirmed that two of them are stilbenoid-specific, advancing our understanding of this specialized enzyme family and shedding critical light onto the biosynthesis of bioactive stilbenoids.

Functional characterization of the vitamin K2 biosynthetic enzyme UBIAD1.

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) plays a significant role in vitamin K2 (MK-4) synthesis. We investigated the enzymological properties of UBIAD1 using microsomal fractions from Sf9 cells expressing UBIAD1 by analysing MK-4 biosynthetic activity. With regard to UBIAD1 enzyme reaction conditions, highest MK-4 synthetic activity was demonstrated under basic conditions at a pH between 8.5 and 9.0, with a DTT ≥0.1 mM. In addition, we found that geranyl pyrophosphate and farnesyl pyrophosphate were also recognized as a side-chain source and served as a substrate for prenylation. Furthermore, lipophilic statins were found to directly inhibit the enzymatic activity of UBIAD1. We analysed the aminoacid sequences homologies across the menA and UbiA families to identify conserved structural features of UBIAD1 proteins and focused on four highly conserved domains. We prepared protein mutants deficient in the four conserved domains to evaluate enzyme activity. Because no enzyme activity was detected in the mutants deficient in the UBIAD1 conserved domains, these four domains were considered to play an essential role in enzymatic activity. We also measured enzyme activities using point mutants of the highly conserved aminoacids in these domains to elucidate their respective functions. We found that the conserved domain I is a substrate recognition site that undergoes a structural change after substrate binding. The conserved domain II is a redox domain site containing a CxxC motif. The conserved domain III is a hinge region important as a catalytic site for the UBIAD1 enzyme. The conserved domain IV is a binding site for Mg2+/isoprenyl side-chain. In this study, we provide a molecular mapping of the enzymological properties of UBIAD1.

A continuous fluorescent assay for protein prenyltransferases measuring diphosphate release.

Protein farnesyltransferase and protein geranylgeranyltransferase type I catalyze the transfer of a 15- and a 20-carbon prenyl group, respectively, from a prenyl diphosphate to a cysteine residue at the carboxyl terminus of target proteins, with the concomitant release of diphosphate. Common substrates include oncogenic Ras proteins, which are implicated in up to 30% of all human cancers, making prenyltransferases a viable target for chemotherapeutic drugs. A coupled assay has been developed to measure the rate constant of diphosphate (PPi) dissociation during the prenyltransferase reaction under both single and multiple turnover conditions. In this assay, the PPi group produced in the prenyltransferase reaction is rapidly cleaved by inorganic pyrophosphatase to form phosphate (Pi), which is then bound by a coumarin-labeled phosphate binding protein from Escherichia coli, resulting in a fluorescence increase. The observed rate constant for PPi release is equal to the rate constant of prenylation of the peptide, as measured by other assays, so that this nonradioactive assay can be used to measure prenyltransferase activity under either single or multiple turnover conditions. This assay can be adapted for high-throughput screening for potential prenyltransferase substrates and inhibitors.

Spectroscopic study of fluorescent peptides for prenyl transferase assays.

A study of the prenyl transferase reactions was performed by fluorescence using rat brain cytosol fractions as an enzyme source. Four dansylated peptides corresponding to the C-terminal sequence of Ras isoforms were synthesised. The effects of different detergents on the farnesylation or geranylgeranylation of the four peptides were evaluated. Dose-dependent effects of dodecyl-maltoside, a non-ionic detergent, on the farnesyl transferase or geranylgeranyl transferase activities were observed with all peptide substrates. Additionally, the effect of temperature was investigated and these assays were applied to determine Michaelis-Menten constants (K(m)) of the substrates: dansyl-GCVLS (1.8 microM), dansyl-GCVVM (3.2 microM), dansyl-CVIM (3.4 microM) and dansyl-GCVLL (8.4 microM) and FPP (22.6 microM) for FTase activity. Using GGPP as co-substrate, GGTase activity was measured with K(m) values superior to 50 microM for all the three substrate dansyl-GCVLS, dansyl-GCVVM, or dansyl-CVIM, whereas values of 7.6 and 5.4 microM were calculated for the dansyl-GCVLL sequence and GGPP co-substrate, respectively. IC50 values of selective prenyl transferase inhibitors, B-581, FTI 276 and GGTI 287 have been measured to 34, 0.8 and 18 nM, respectively, using dansyl-GCVLS as substrate (FTase inhibition). When dansyl-GCVLL is used as substrate (GGTase inhibition) the IC50 values are 5100, 75 and 5 nM for B-581, FTI 276 and GGTI 287, respectively. Then, this developed method allowed to evaluate the selectivity of all the three inhibitors tested.

Interconversion of the product specificity of type I eubacterial farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase through one amino acid substitution.

Prenyltransferases catalyze the sequential condensation of isopentenyl diphosphate into prenyl diphosphates with specific chain lengths. Pioneering studies demonstrated that the product specificities of type I prenyltransferases were mainly determined by the amino acid residues at the 4th and 5th positions before the first aspartate-rich motif (FARM) of the prenyltransferases. We previously cloned a type I geranylgeranyl diphosphate synthase (GGDPSase) gene from Streptomyces griseolosporeus MF730-N6 [Hamano, Y., Dairi, T., Yamamoto, M., Kawasaki, T., Kaneda, K., Kuzuyama, T., Itoh, N., and Seto, H. (2001) BIOSCI: Biotechnol. Biochem. 65, 1627-1635]. In this study, a prenyltransferase gene was cloned from Streptomyces argenteolus A-2 and was confirmed to encode a type I farnesyl diphosphate synthase (FDPSase). Interestingly, the amino acid residues at the 4th and 5th positions before the FARM were the same in these two enzymes. To identify the amino acid that determines the product chain length, mutated enzymes, GGDPSase (L-50S), FDPSase (S-50L), GGDPSase (V-8A), FDPSase (A-8V), GGDPSase (A+57L), and FDPSase (L+58A), in which the amino acid residue at the -50th, -8th, and +57th (58th) position before or after the FARM was substituted with the corresponding amino acid of the other enzyme, were constructed. The GGDPSase (A+57L) and FDPSase (L+58A) produced farnesyl diphosphate and geranylgeranyl diphosphate, respectively. On the other hand, the other mutated enzymes produced prenyl diphosphates with the same chain lengths as the wild type enzymes did. These results showed that the amino acid residue at the 57th (58th) position after the FARM also played an important role in determination of the product specificity.

Biosynthesis of prenyl diphosphates by cell-free extracts from mammalian tissues.

When assayed by the conventional method for prenyltransferase using a combination of [1-14C]isopentenyl and geranyl diphosphates, 100,000 x g supernatants of homogenates of rat liver and brain catalyzed the formation of geranylgeranyl diphosphate at a much lower rate than that of farnesyl diphosphate. Surprisingly, however, the formation of geranylgeranyl diphosphate in incubations of [1-14C]isopentenyl diphosphate alone with these enzyme systems was comparable to that of farnesyl diphosphate. Addition of dimethylallyl diphosphate to the same enzyme systems in the presence of [1-14C]isopentenyl diphosphate resulted in a marked increase in the rate of formation of farnesyl diphosphate, while the rate of formation of geranylgeranyl diphosphate was saturated. Metabolic labeling of rat liver and kidney slices with [5-3H]mevalonic acid revealed that the major prenyl residue of the detectable prenylated proteins was actually the geranylgeranyl group. Coupled with the previous finding that geranylgeranyl diphosphate accumulates during metabolic labeling of rat liver slices with [2-3H]mevalonic acid [Sagami, H., Matsuoka, S., and Ogura, K. (1991) J. Biol. Chem. 266, 3458-3463], these results indicate that the rate of de novo synthesis of geranylgeranyl diphosphate from mevalonic acid is comparable to that of farnesyl diphosphate.

Occurrence of the chromoplast protein ChrA correlates with a fruit-color gene in Capsicum annuum.

Plant geneticists have determined that the color of ripe fruits of sweet peppers (Capsicum annuum L.) is determined by four genes: y, c1, c2 and cl. We have compared the electrophoretic behavior of chromoplast membrane proteins of seven varieties of C. annuum which differ in these genes. ChrA was detected only in the varieties that had a y+ genotype, and was not affected by variations in the other three genes. The identity of ChrA was verified by probing blots of SDS gels with antiserum to ChrA. The second known chromoplast-specific protein, ChrB, was found to be independent of all four genes. No proteins correlating with c1, c2 or cl were detected in either one- or two-dimensional gels.

Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase.

Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.

Hydrophobic chromatography of prenyl transferases.

Several prenyl transferases were examined with respect to their affinity for C0--C10 alkyl-Agarose columns. C10-alkyl Agarose was effective in adsorbing each of the prenyl transferases tested but only undecaprenyl pyrophosphate and octaprenyl pyrophosphate synthetases could be effectively eluted with Triton X-100.