Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution.

In the synthesis of isoprenoids, isoprenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce a variety of prenyl diphosphates with well-defined chain lengths. Site-directed mutagenesis in conjunction with X-ray crystallographic studies have identified specific amino acid residues responsible for chain-length determination. Simple combinations of these residues within a characteristic motif are not only sufficient to confer product specificities to all isoprenyl diphosphate synthases but represent structural features that reflect the enzyme family's evolutionary course.     

A thermophilic cyanobacterium Synechococcus elongatus has three different Class I prenyltransferase genes

Prenyltransferases (prenyl diphosphate synthases), which are a broad group of enzymes that catalyze the consecutive condensation of homoallylic diphosphate of isopentenyl diphosphates (IPP, C₅) with allylic diphosphates to synthesize prenyl diphosphates of various chain lengths, have highly conserved regions in their amino acid sequences. Based on the above information, three prenyltransferase homologue genes were cloned from a thermophilic cyanobacterium, Synechococcus elongatus. Through analyses of the reaction products of the enzymes encoded by these genes, it was revealed that one encodes a thermolabile geranylgeranyl (C₂₀) diphosphate synthase, another encodes a farnesyl (C₁₅) diphosphate synthase whose optimal reaction temperature is 60 °C, and the third one encodes a prenyltransferase whose optimal reaction temperature is 75 °C. The last enzyme could catalyze the synthesis of five prenyl diphosphates of farnesyl, geranylgeranyl, geranylfarnesyl (C₂₅), hexaprenyl (C₃₀), and heptaprenyl (C₃₅) diphosphates from dimethylallyl (C₅) diphosphate, geranyl (C₂₀) diphosphate, or farnesyl diphosphate as the allylic substrates. The product specificity of this novel kind of enzyme varied according to the ratio of the allylic and homoallylic substrates. The situations of these three S. elongatus enzymes in a phylogenetic tree of prenyltransferases are discussed in comparison with a mesophilic cyanobacterium of Synechocystis PCC6803, whose complete genome has been reported by Kaneko et al. (1996).     

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechanism of chain length determination in type III geranylgeranyl diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the alpha-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase.     

Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Two open reading frames which encode the homologues of (all-E) prenyl diphosphate synthase are found in the whole-genome sequence of Sulfolobus solfataricus, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in Escherichia coli cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg(2+) and Triton X-100 for full activity. It catalyzes consecutive E-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.     

Mechanism of product chain length determination for heptaprenyl diphosphate synthase from Bacillus stearothermophilus.

A member of the medium-chain prenyl diphosphate synthases, Bacillus stearothermophilus heptaprenyl diphosphate synthase, catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphate to produce (all-E)-C35 prenyl diphosphate as the ultimate product. We previously showed that the product specificity of short-chain prenyl diphosphate synthases is regulated by the structure around the first aspartate-rich motif (FARM). The FARM is also conserved in a subunit of heptaprenyl diphosphate synthase, component II', which suggests that the structure around the FARM of component II' regulates the elongation. To determine whether component II' regulates the product chain length by a mode similar to that of the short-chain prenyl diphosphate synthases, we replaced a bulky amino acid at the eighth position before the FARM of component II', isoleucine 76, by glycine and analyzed the product specificity. The mutated enzyme, I76G, can catalyze condensations of isopentenyl diphosphate beyond the native chain length of C35. Moreover, two mutated enzymes of A79Y and S80F, which have a single replacement to the aromatic residue at the fourth or the fifth position before the FARM, mainly yielded a C20 product. These results strongly suggest that a common mechanism controls the product chain length of both short-chain and medium-chain prenyl diphosphate synthases and that, in wild-type heptaprenyl diphosphate synthase, the prenyl chain can grow on the surface of the small residues at positions 79 and 80, and the elongation is precisely blocked at the length of C35 by isoleucine 76.     

Artificial substrates of medium-chain elongating enzymes, hexaprenyl- and heptaprenyl diphosphate synthases

We examined the reactivity of 3-alkyl group homologues of farnesyl diphosphate or isopentenyl diphosphate for medium-chain prenyl diphosphate synthases, hexaprenyl diphosphate- or heptaprenyl diphosphate synthase. But-3-enyl diphosphate, which lacks the methyl group at the 3-position of isopentenyl diphosphate, condensed only once with farnesyl diphosphate to give E-norgeranylgeranyl diphosphate by the action of either enzyme. However, norfarnesyl diphosphate was never accepted as an allylic substrate at all. 3-Ethylbut-3-enyl diphosphate also reacted with farnesyl diphosphate giving a mixture of (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all-E)-3,7-diethyl-11,15,19-trimethylicosa-2,6,10,14,18-pentaenyl diphosphates by hexaprenyl diphosphate synthase. On the other hand, heptaprenyl diphosphate synthase reaction of 3-ethylbut-3-enyl diphosphate with farnesyl diphosphate gave only (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate.     

Chain length determination of prenyltransferases: both heteromeric subunits of medium-chain (E)-prenyl diphosphate synthase are involved in the product chain length determination

Among prenyltransferases, medium-chain (E)-prenyl diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638-14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase and Bacillus subtilis heptaprenyl diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major products, and a double combination of mutant subunits of the heptaprenyl diphosphate synthase, I-D97A/II-A79F, yielded exclusively geranylgeranyl diphosphate. However, the combination of a mutant subunit and the wild-type, I-Y103S/II-WT or I-WT/II-I76G, produced a C(40) prenyl diphosphate, and the double combination of the mutants, I-Y103S/II-I76G, gave a reaction product with longer prenyl chain up to C(50). These results suggest that medium-chain (E)-prenyl diphosphate synthases take a novel mode for the product chain length determination, in which both subunits cooperatively participate in maintaining and determining the product specificity of each enzyme.     

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.     

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.     

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.     

Chain elongation in the isoprenoid biosynthetic pathway

Isoprenyl diphosphate synthases catalyze addition of allylic diphosphate primers to the isoprene unit in isopentenyl diphosphate to produce polyisoprenoid diphosphates with well defined chain lengths. Phylogenetic correlations suggest that the synthases which catalyze formation of isoprenoid diphosphates with (E) double bonds have evolved from a common ancestor. X-ray crystallographic studies of farnesyl diphosphate synthase in conjunction with site-directed mutagenesis have provided important new information about the residues involved in binding and catalysis and the source of chain length selectivity for the enzymes that catalyze chain elongation.     

Geranyl diphosphate synthase from Abies grandis: cDNA isolation,functional expression,and characterization

Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.     

Decaprenyl diphosphate synthesis in Mycobacterium tuberculosis

Z-prenyl diphosphate synthases catalyze the sequential condensation of isopentenyl diphosphate with allylic diphosphates to synthesize polyprenyl diphosphates. In mycobacteria, these are precursors of decaprenyl phosphate, a molecule which plays a central role in the biosynthesis of essential mycobacterial cell wall components, such as the mycolyl-arabinogalactan-peptidoglycan complex and lipoarabinomannan. Recently, it was demonstrated that open reading frame Rv2361c of the Mycobacterium tuberculosis H37Rv genome encodes a unique prenyl diphosphate synthase (M. C. Schulbach, P. J. Brennan, and D. C. Crick, J. Biol. Chem. 275:22876-22881, 2000). We have now purified the enzyme to near homogeneity by using an Escherichia coli expression system and have shown that the product of this enzyme is decaprenyl diphosphate. Rv2361c has an absolute requirement for divalent cations and an optimal pH range of 7.5 to 8.5, and the activity is stimulated by both detergent and dithiothreitol. The enzyme catalyzes the addition of isopentenyl diphosphate to geranyl diphosphate, neryl diphosphate, omega,E,E-farnesyl diphosphate, omega,E,Z-farnesyl diphosphate, or omega,E,E,E-geranylgeranyl diphosphate, with Km values for the allylic substrates of 490, 29, 84, 290, and 40 microM, respectively. The Km value for isopentenyl diphosphate is 89 microM. The catalytic efficiency is greatest when omega,E,Z-farnesyl diphosphate is used as the allylic acceptor, suggesting that this is the natural substrate in vivo, a conclusion that is supported by previous structural studies of decaprenyl phosphoryl mannose isolated from M. tuberculosis. This is the first report of a bacterial Z-prenyl diphosphate synthase that preferentially utilizes an allylic diphosphate primer having the alpha-isoprene unit in the Z configuration, indicating that Rv1086 (omega,E,Z-farnesyl diphosphate synthase) and Rv2361c act sequentially in the biosynthetic pathway that leads to the formation of decaprenyl phosphate in M. tuberculosis.     

Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.     

Arabidopsis thaliana isoprenyl diphosphate synthases produce the C25 intermediate geranylfarnesyl diphosphate

Isoprenyl diphosphate synthases (IDSs) catalyze some of the most basic steps in terpene biosynthesis by producing the prenyl diphosphate precursors of each of the various terpenoid classes. Most plants investigated have distinct enzymes that produce the short‐chain all‐trans (E) prenyl diphosphates geranyl diphosphate (GDP, C₁₀), farnesyl diphosphate (FDP, C₁₅) or geranylgeranyl diphosphate (GGDP, C₂₀). In the genome of Arabidopsis thaliana, 15 trans‐product‐forming IDSs are present. Ten of these have recently been shown to produce GGDP by genetic complementation of a carotenoid pathway engineered into Escherichia coli. When verifying the product pattern of IDSs producing GGDP by a new LC‐MS/MS procedure, we found that five of these IDSs produce geranylfarnesyl diphosphate (GFDP, C₂₅) instead of GGDP as their major product in enzyme assays performed in vitro. Over‐expression of one of the GFDP synthases in A. thaliana confirmed the production of GFDP in vivo. Enzyme assays with A. thaliana protein extracts from roots but not other organs showed formation of GFDP. Furthermore, GFDP itself was detected in root extracts. Subcellular localization studies in leaves indicated that four of the GFDP synthases were targeted to the plastoglobules of the chloroplast and one was targeted to the mitochondria. Sequence comparison and mutational studies showed that the size of the R group of the 5th amino acid residue N‐terminal to the first aspartate‐rich motif is responsible for C₂₅ versus C₂₀ product formation, with smaller R groups (Ala and Ser) resulting in GGDP (C₂₀) as a product and a larger R group (Met) resulting in GFDP (C₂₅).     

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity

Directed evolution of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme. The 12 evolved clones having enhanced C20 GGPP production have characteristic mutations in the conserved regions of prenyl diphosphate synthases (designated regions I through VII). Some of these mutations (I76T, Y79S, Y79H, C75Y, H83Y, and H83Q) are found near or before the conserved first aspartate rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl synthases. Molecular modeling suggested a mechanism for chain length determination for these mutations including substitutions at the 1st and 9th amino acids upstream of the FARM that have not been reported previously. In addition, a mutation on a helix adjacent to the FARM within the substrate-binding pocket (D115G) suggests a novel mechanism for chain length determination. One mutant IspA clone carries a mutation of C155G at the 2nd amino acid upstream of conserved region IV (GQxxDL), which was recently found to be an important region controlling the chain elongation of a Type III GGPP synthase. One IspA clone carries mutations (T234A and T249I) near the conserved second aspartate rich motif (SARM). As a verification of the in vivo activity of the mutant clones (represented as C40 carotenoid formation), we confirmed the product distribution of wild-type and mutant IspA using an in vitro assay.     

Biochemical and structural studies with prenyl diphosphate analogues provide insights into isoprenoid recognition by protein farnesyl transferase

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.     

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.     

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (-)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.