Geranylgeranyl diphosphate synthases from Scoparia dulcis and Croton sublyratus. cDNA cloning, functional expression, and conversion to a farnesyl diphosphate synthase

cDNAs encoding geranylgeranyl diphosphate synthase (GGPPS) of two diterpene producing plants, Scoparia dulcis and Croton sublyratus, were isolated using the homology-based polymerase chain reaction method. Both cloned genes showed high amino acid sequence homology (60-70%) to other plant GGPPSs and contained highly conserved aspartate-rich motifs. The obtained clones were functionally expressed in Escherichia coli and showed sufficient GGPPS activity to catalyze the condensation of farnesyl diphosphate (FPP) and isopentenyl diphosphate to form geranylgeranyl diphosphate. To investigate the factor determining the product chain length of plant GGPPSs, S. dulcis GGPPS mutants in which either the small amino acids at the fourth and fifth positions before the first aspartate-rich motif (FARM) were replaced with aromatic amino acids or in which two additional amino acids in FARM were deleted were constructed. Both mutants behaved like FPPS-like enzymes and almost exclusively produced FPP when dimethylallyl diphosphate was used as a primer substrate, and failed to accept FPP as a primer substrate. These results indicate that both small amino acids at the fourth and fifth positions before FARM and the amino acid insertion in FARM play essential roles in product length determination in plant GGPPSs.     

Bifunctional abietadiene synthase: free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinct cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosynthesis in grand fir (Abies grandis). The first reaction converts geranylgeranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphosphate via protonation-initiated cyclization. In the second reaction, diphosphate ester ionization-initiated cyclization generates the tricyclic perhydrophenanthrene-type backbone, and is directly coupled to a 1,2-methyl migration that generates the C13 isopropyl group characteristic of the abietane family of diterpenes. Using the transition-state analogue inhibitor 14,15-dihydro-15-azageranylgeranyl diphosphate, it was demonstrated that each reaction of abietadiene synthase is carried out at a distinct active site. Mutations in two aspartate-rich motifs specifically delete one or the other activity and the location of these motifs suggests that the two active sites reside in separate domains. These mutants effectively complement each other, suggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detected in steady-state kinetic reactions, thus conclusively demonstrating a free diffusion transfer mechanism. In addition, both mutant enzymes enhance the activity of wild-type abietadiene synthase with geranylgeranyl diphosphate as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.     

Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.     

Chrysanthemyl diphosphate synthase. The relationship among chain elongation,branching, and cyclopropanation reactions in the isoprenoid biosynthetic pathway

The genes for chrysanthemyl diphosphate (CPP) synthase and farnesyl diphosphate (FPP) synthase from sagebrush, Artemisia tridentata spiciformis, were used to prepare a series of chimeric proteins to investigate the 1'-4 chain elongation, 1'-2 branching, and c1'-2-3 cyclopropanation reactions that join isoprenoid units to build more complex structures. The two genes were modified by site-directed mutagenesis to generate an identical set of six unique restriction sites at identical locations. The locations were selected to place a restriction site between each of the five conserved regions found in prenyltransferases that catalyze chain elongation. A series of chimeric proteins were generated by replacing amino acids in FPP synthase, beginning at the N-terminus of the enzyme, with increasing stretches of peptide from CPP synthase. An analysis of the products produced by the chimeras revealed a transition from 1'-4 chain elongation, to 1'-2 branching, and ultimately to c1'-2-3 cyclopropanation. These results demonstrate that the catalytic site for chain elongation, with minor modifications in its architecture, also catalyzes 1'-2 branching and c1'-2-3 cyclopropanation, and suggest that the branching and cyclopropanation reactions, in analogy to chain elongation, are electrophilic alkylations.     

Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors

Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPP(i)) was prepared from trimethyl phosphate in four steps. The tris(tetra-n-butylammonium) salt was then used to convert isopentenyl tosylate to (S)-isopentenyl thiodiphosphate (ISPP). (S)-Dimethylallyl (DMASPP), (S)-geranyl (GSPP), (S)-farnesyl (FSPP), and (S)-geranylgeranyl thiodiphosphate (GGSPP) were prepared from the corresponding bromides in a similar manner. ISPP and GSPP were substrates for avian farnesyl diphosphate synthase (FPPase). Incubation of the enzyme with ISPP and GPP gave FSPP, whereas incubation with IPP and GSPP gave FPP. GSPP was a substantially less reactive than GPP in the chain elongation reaction and was an excellent competitive inhibitor, K(I)(GSPP) = 24.8 microM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.     

Pentalenene synthase. Analysis of active site residues by site-directed mutagenesis

Incubation of farnesyl diphosphate (1) with the W308F or W308F/H309F mutants of pentalenene synthase, an enzyme from Streptomyces UC5319, yielded pentalenene (2), accompanied by varying proportions of (+)-germacrene A (7) with relatively minor changes in k(cat) and k(cat)/K(m). By contrast, single H309 mutants gave rise to both (+)-germacrene A (7) and protoilludene (8) in addition to pentalenene (2). Mutation to glutamate of each of the three aspartate residues in the Mg(2+)-binding aspartate-rich domain, (80)DDLFD, resulted in reduction in the k(cat)/K(m) for farnesyl diphosphate and formation of varying proportions of pentalenene and (+)-germacrene A (7). Formation of (+)-germacrene A (7) by the various pentalenene synthase mutants is the result of a derailment of the natural anti-Markovnikov cyclization reaction, and not simply the consequence of trapping of a normally cryptic, carbocationic intermediate. Both the N219A and N219L mutants of pentalenene synthase were completely inactive, while the corresponding N219D mutant had a k(cat)/K(m) which was 3300-fold lower than that of the wild-type synthase, and produced a mixture of pentalenene (2) (91%) and the aberrant cyclization product beta-caryophyllene (9) (9%). Finally, the F77Y mutant had a k(cat)/K(m) which was reduced by 20-fold compared to that of the wild-type synthase.     

Geranylgeraniol formation in Croton stellatopilosus proceeds via successive monodephosphorylations of geranylgeranyl diphosphate

The process of catalytic dephosphorylation of geranylgeranyl diphosphate (GGPP) to give geranylgeraniol (GGOH) in Croton stellatopilosus leaves was examined by in vivo chloroplast feedings with [1-³H]GGPP and [1-³H]GGMP and in vitro enzyme-catalyzed reactions. The results strongly suggest that the formation of GGOH from GGPP proceeds in the chloroplasts via two successive monodephosphorylation reactions. Hence, we name the enzyme geranylgeranyl diphosphate phosphatase rather than geranylgeranyl diphosphatase based on its catalytic mechanism.     

Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin

Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.     

A soluble form of phosphatase in Saccharomyces cerevisiae capable of converting farnesyl diphosphate into E,E-farnesol

After anion-exchange chromatography, the soluble fraction of a cell-free extract of Saccharomyces cerevisiae showed two phosphatase activity peaks when p-nitrophenyl phosphate (pNPP) was used as the substrate. However, only the second pNPP active peak demonstrated the ability to convert farnesyl diphosphate (FPP) into E,E-farnesol. N-terminal sequence analysis of the purified pNPP/FPP phosphatase revealed that it was a truncated form of alkaline phosphatase Pho8 lacking 62 amino acids from the N-terminus and was designated Pho8Delta62. Although other isoprenyl diphosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) could also be hydrolyzed by Pho8Delta62 to the corresponding alcohols, selectivity was observed among these substrates. The optimum pH was 7.0 for all three isoprenyl diphosphate substrates. Although lower hydrolytic activity was observed for FPP and GGPP at pH 6.0 and 8.5, hydrolysis of GPP was observed only at pH 7.0. Mg2+ and Mn2+ inhibited hydrolysis of FPP and GGPP, and GGPP was more sensitive to Mg2+ inhibition than FPP. The rate of FPP hydrolysis increased in the presence of Triton X-100.     

Genome mining in Streptomyces coelicolor: molecular cloning and characterization of a new sesquiterpene synthase

The terpene synthase encoded by the SCO5222 (SC7E4.19) gene of Streptomyces coelicolor was cloned by PCR and expressed in Escherichia coli as an N-terminal-His6-tag protein. Incubation of the recombinant protein, SCO5222p, with farnesyl diphosphate (1, FPP) in the presence of Mg(II) gave a new sesquiterpene, (+)-epi-isozizaene (2), whose structure and stereochemistry were determined by a combination of 1H, 13C, COSY, HMQC, HMBC, and NOESY NMR. The steady-state kinetic parameters were kcat 0.049 +/- 0.001 s-1 and a Km (FPP) of 147 +/- 14 nM. Individual incubations of recombinant epi-isozizaene synthase with [1,1-2H2]FPP (1a), (1R)-[1-2H]-FPP (1b), and (1S)-[1-2H]-FPP (1c) and NMR analysis of the resulting deuterated epi-isozizaenes supported an isomerization-cyclization-rearrangement mechanism involving the intermediacy of (3R)-nerolidyl diphosphate (3).     

A common mechanism for branching, cyclopropanation, and cyclobutanation reactions in the isoprenoid biosynthetic pathway

Four reactions--chain elongation, cyclopropanation, branching, and cyclobutanation--are used in nature to join isoprenoid units for construction of the carbon skeletons for over 55,000 naturally occurring isoprenoid compounds. Those molecules produced by chain elongation have head-to-tail (regular) carbon skeletons, while those from cyclopropanation, branching, or cyclobutanation have non-head-to-tail (irregular) skeletons. Although wild type enzymes have not been identified for the branching and cyclobutanation reactions, chimeric proteins constructed from farnesyl diphosphate synthase (chain elongation) and chrysanthemyl diphosphate synthase (cyclopropanation) catalyze all four of the known isoprenoid coupling reactions to give a mixture of geranyl diphosphate (chain elongation), chrysanthemyl diphosphate (cyclopropanation), lavandulyl diphosphate (branching), and maconelliyl and planococcyl diphosphate (cyclobutanation). Replacement of the hydrogen atoms at C1 or C2 or hydrogen atoms in the methyl groups of dimethylallyl diphosphate by deuterium alters the distribution of the cyclopropanation, branching, and cyclobutanation products through primary and secondary kinetic isotope effects on the partitioning steps of common carbocationic intermediates. These experiments establish the sequence in which the intermediates are formed and indicate that enzyme-mediated control of the carbocationic rearrangement and elimination steps determines the distribution of products.     

Recombinant squalene synthase. A mechanism for the rearrangement of presqualene diphosphate to squalene

Squalene synthase (SQase) catalyzes the condensation of two molecules of farnesyl diphosphate (FPP) to form presqualene diphosphate (PSPP) and the subsequent rearrangement and NADPH-dependent reduction of PSPP to squalene (SQ). These reactions are the first committed steps in cholesterol biosynthesis. When recombinant SQase was incubated with FPP in the presence of dihydroNADPH (NADPH3, an unreactive analogue lacking the 5,6-double bond in the nicotinamide ring), three products were formed: dehydrosqualene (DSQ), a C30 analogue of phytoene; 10(S)-hydroxysqualene (HSQ), a hydroxy analogue of squalene; and rillingol (ROH), a cyclopropylcarbinyl alcohol formed by addition of water to the tertiary cyclopropylcarbinyl cation previously proposed as an intermediate in the rearrangement of PSPP to SQ (Poulter, C. D. Acc. Chem. Res. 1990, 23, 70-77). The structure and absolute stereochemistry of the tertiary cyclopropylcarbinyl alcohol were established by synthesis using two independent routes. Isolation of ROH from the enzyme-catalyzed reaction provides strong evidence for a cyclopropylcarbinyl-cyclopropylcarbinyl rearrangement in the biosynthesis of squalene. By comparing the SQase-catalyzed solvolysis of PSPP in the absence of NADPH3 to the reaction in the presence of NADPH3, it is apparent that the binding of the cofactor analogue substantially enhances the ability of SQase to control the regio- and stereochemistry of the rearrangements of PSPP.     

Farnesyl diphosphate synthase: the art of compromise between substrate selectivity and stereoselectivity

Farnesyl diphosphate (FPP) synthase catalyzes the consecutive head-to-tail condensations of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to give (E,E)-FPP (C15). The enzyme belongs to a genetically distinct family of chain elongation enzymes that install E-double bonds during each addition of a five-carbon isoprene unit. Analysis of the C10 and C15 products from incubations with avian FPP synthase reveals that small amounts of neryl diphosphate (Z-C10) and (Z,E)-FPP are formed along with the E-isomers during the C5 --> C10 and C10 --> C15 reactions. Similar results were obtained for FPP synthase from Escherichia coli, Artemisia tridentata (sage brush), Pyrococcus furiosus, and Methanobacter thermautotrophicus and for GPP and FPP synthesized in vivo by E. coli FPP synthase. When (R)-[2-2H]IPP was a substrate for chain elongation, no deuterium was found in the chain elongation products. In contrast, the deuterium in (S)-[2-2H]IPP was incorporated into all of the products. Thus, the pro-R hydrogen at C2 of IPP is lost when the E- and Z-double bond isomers are formed. The synthesis of Z-double bond isomers by FPP synthase during chain elongation is unexpected for a highly evolved enzyme and probably reflects a compromise between optimizing double bond stereoselectivity and the need to exclude DMAPP from the IPP binding site.     

Intramolecular proton transfer in the cyclization of geranylgeranyl diphosphate to the taxadiene precursor of taxol catalyzed by recombinant taxadiene synthase

BACKGROUND: The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers. RESULTS: Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5. CONCLUSIONS: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes.     

Aristolochene synthase: mechanistic analysis of active site residues by site-directed mutagenesis

Incubation of farnesyl diphosphate (1) with Penicillium roqueforti aristolochene synthase yielded (+)-aristolochene (4), accompanied by minor quantities of the proposed intermediate (S)-(-)germacrene A (2) and the side-product (-)-valencene (5) in a 94:4:2 ratio. By contrast, the closely related aristolochene synthase from Aspergillus terreus cyclized farnesyl diphosphate only to (+)-aristolochene (4). Site-directed mutagenesis of amino acid residues in two highly conserved Mg(2+)-binding domains led in most cases to reductions in both k(cat) and k(cat)/K(m) as well as increases in the proportion of (S)-(-)germacrene A (2), with the E252Q mutant of the P. roqueforti aristolochene synthase producing only (-)-2. The P. roqueforti D115N, N244L, and S248A/E252D mutants were inactive, as was the A. terreus mutant E227Q. The P. roqueforti mutant Y92F displayed a 100-fold reduction in k(cat) that was offset by a 50-fold decrease in K(m), resulting in a relatively minor 2-fold decrease in catalytic efficiency, k(cat)/K(m). The finding that Y92F produced (+)-aristolochene (4) as 81% of the product, accompanied by 7% 5 and 12% 2, rules out Tyr-92 as the active site Lewis acid that is responsible for protonation of the germacrene A intermediate in the formation of aristolochene (4).     

A soluble form of phosphatase in Saccharomyces cerevisiae capable of converting farnesyl diphosphate into E,E-farnesol

After anion-exchange chromatography, the soluble fraction of a cell-free extract of Saccharomyces cerevisiae showed two phosphatase activity peaks when p-nitrophenyl phosphate (pNPP) was used as the substrate. However, only the second pNPP active peak demonstrated the ability to convert farnesyl diphosphate (FPP) into E,E-farnesol. N-terminal sequence analysis of the purified pNPP/FPP phosphatase revealed that it was a truncated form of alkaline phosphatase Pho8 lacking 62 amino acids from the N-terminus and was designated Pho8Δ62. Although other isoprenyl diphosphates such as geranyl diphosphate (GPP) and geranylgeranyl diphosphate (GGPP) could also be hydrolyzed by Pho8Δ62 to the corresponding alcohols, selectivity was observed among these substrates. The optimum pH was 7.0 for all three isoprenyl diphosphate substrates. Although lower hydrolytic activity was observed for FPP and GGPP at pH 6.0 and 8.5, hydrolysis of GPP was observed only at pH 7.0. Mg²⁺ and Mn²⁺ inhibited hydrolysis of FPP and GGPP, and GGPP was more sensitive to Mg²⁺ inhibition than FPP. The rate of FPP hydrolysis increased in the presence of Triton X-100.     

Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase

Bisphosphonates, known for their effectiveness in the treatment of osteoporosis, inhibit bone resorption via mechanisms that involve binding to bone mineral and cellular effects on osteoclasts. The major molecular target of nitrogen-containing bisphosphonates (N-BPs) in osteoclasts is farnesyl diphosphate synthase (FPPS). N-BPs likely inhibit this enzyme by mimicking one or more of the natural isoprenoid lipid substrates (GPP/DMAPP and IPP) but the mode of inhibition is not established. The active site of FPPS comprises a subsite for each substrate. Kinetic studies with recombinant human FPPS indicate that both potent (risedronate) and weak (NE-58051) enzyme inhibitors compete with GPP for binding to FPPS, however, binding to this site does not completely explain the difference in potency of the two inhibitors, suggesting that a second binding site may also be a target of bisphosphonate inhibition. Using the docking software suite Autodock, we explored a dual inhibitor binding mode for recombinant human FPPS. Experimental support for dual binding is suggested by Dixon plots for the inhibitors. N-BPs may inhibit by binding to both the GPP and a second site with differences in potency at least partly arising from inhibition at the second site.     

Farnesyl diphosphate analogues with omega-bioorthogonal azide and alkyne functional groups for protein farnesyl transferase-catalyzed ligation reactions

Eleven farnesyl diphosphate analogues, which contained omega-azide or alkyne substituents suitable for bioorthogonal Staudinger and Huisgen [3 + 2] cycloaddition coupling reactions, were synthesized. The analogues were evaluated as substrates for the alkylation of peptide cosubstrates by yeast protein farnesyl transferase. Five of the diphosphates were good alternative substrates for farnesyl diphosphate (FPP). Steady-state kinetic constants were measured for the active compounds, and the products were characterized by HPLC and LC-MS. Two of the analogues gave steady-state kinetic parameters (kcat and Km) very similar to those of the natural substrate.     

Recombinant squalene synthase. Synthesis of non-head-to-tail isoprenoids in the absence of NADPH

Squalene synthase (SQase) catalyzes two consecutive reactions in sterol biosynthesis. The first is the condensation of two molecules of farnesyl diphosphate (FPP) to form a cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). The subsequent conversion of PSPP to squalene (SQ) involves an extensive rearrangement of the carbon skeleton and a NADPH-dependent reduction. Incubation of a truncated soluble form of recombinant yeast SQase with FPP in buffer lacking NADPH gave (1R,2R,3R)-PSPP. As the incubation continued, SQase catalyzed the subsequent conversion of PSPP to a mixture of triterpenes. Two of the major products, (Z)-dehydrosqualene (DSQ) and (R)-12-hydroxysqualene (HSQ), have the same 1'-1 linkage between the farnesyl units from FPP that is found in squalene. The other major product, (10S,13S)-10-hydroxybotryococcene (HBO), has a 1'-3 linkage between the farnesyl units. Small quantities of (S)-HSQ and (10R,13S)-HBO were also formed. Three additional triterpenes, the allylic isomers of HSQ and HBO, and an unidentified alcohol were produced in minor amounts. A methyl ether corresponding to HSQ was detected when methanol was present in the incubation buffer. These compounds are the expected "solvolysis" products from PSPP. They provide strong support for mechanisms that propose cyclopropylcarbinyl cations as intermediates in the SQase-catalyzed rearrangement of PSPP to SQ and unambiguously demonstrate that the catalytic machinery of SQase is capable of synthesizing a variety of irregular isoprenoids.     

Templating effects in aristolochene synthase catalysis: elimination versus cyclisation

Analysis of the products generated by mutants of aristolochene synthase from P. roqueforti (PR-AS) revealed the prominent structural role played by the aliphatic residue Leu 108 in maintaining the productive conformation of farnesyl diphosphate to ensure C1-C10 (σ-bond) ring-closure and hence (+)-aristolochene production.