Studies on taxadiene synthase: interception of the cyclization cascade at the isocembrene stage with GGPP analogues

[reaction: see text] The cyclization of GGPP to taxadiene, catalyzed by taxadiene synthase, has been suggested to proceed through a series of monocyclic isocembrenyl- and bicyclic verticillyl-carbocationic intermediary stages. A set of GGPP analogues with abolished or perturbed pi-nucleophilicity at the delta10 double bond (GGPP numbering) was synthesized and incubated with taxadiene synthase to intercept the cyclization cascade at the monocyclic stage. Each analogue was transformed by taxadiene synthase in vitro to hydrocarbon products in varying yields, and the structures of the major product in each reaction were solved by GCEIMS and one- and two-dimensional (1H and 13C) NMR and found to be 14-membered monocyclic isocembrenyl diterpenes, indicating that the first C-C bond formation catalyzed by taxadiene synthase could be uncoupled from the other subsequent bond formation events by using suitably designed substrate analogues. The formation and isolation of these isocembrenyl diterpene products using taxadiene synthase supports proposals that the isocembrenyl cation is an intermediate in the cyclization of GGPP to taxadiene.     

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.     

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.     

Enzymatic Formation of a Skipped Methyl‐Substituted Octaprenyl Side Chain of Longestin (KS‐505a): Involvement of Homo‐IPP as a Common Extender Unit

Longestin (KS‐505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo‐IPP) to afford (3Z)‐3‐methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.     

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.     

Theoretical and Experimental Analysis of the Reaction Mechanism of MrTPS2, a Triquinane‐Forming Sesquiterpene Synthase from Chamomile

Terpene synthases, as key enzymes of terpene biosynthesis, have garnered the attention of chemists and biologists for many years. Their carbocationic reaction mechanisms are responsible for the huge variety of terpene structures in nature. These mechanisms are amenable to study by using classical biochemical approaches as well as computational analysis, and in this study we combine quantum‐chemical calculations and deuterium‐labeling experiments to elucidate the reaction mechanism of a triquinane forming sesquiterpene synthase from chamomile. Our results suggest that the reaction from farnesyl diphosphate to triquinanes proceeds through caryophyllyl and presilphiperfolanyl cations and involves the protonation of a stable (?)‐(E)‐β‐caryophyllene intermediate. A tyrosine residue was identified that appears to be involved in the proton‐transfer process.     

QM/MM study of the taxadiene synthase mechanism

Combined quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the reaction mechanism of taxadiene synthase (TXS). TXS catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to taxadiene (T) and four minor cyclic products. All these products originate from the deprotonation of carbocation intermediates. The reaction profiles for the conversion of GGPP to T as well as to minor products were calculated for different configurations of relevant TXS carbocation complexes. The QM region was treated at the M06-2X/TZVP level, while the CHARMM27 force field was used to describe the MM region. The QM/MM calculations suggest a reaction pathway for the conversion of GGPP to T, which slightly differs from previous proposals regarding the number of reaction steps and the conformation of the carbocations. The QM/MM results also indicate that the formation of minor products via water-assisted deprotonation of the carbocations is highly exothermic, by about −7 to −23 kcal/mol. Curiously, however, the computed barriers and reaction energies indicate that the formation of some of the minor products is more facile than the formation of T. Thus, the present QM/MM calculations provide detailed insights into possible reaction pathways and into the origin of the promiscuity of TXS, but they do not reproduce the product distribution observed experimentally. © 2019 Wiley Periodicals, Inc.     

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.     

Pristinol,a Sesquiterpene Alcohol with an Unusual Skeleton from Streptomyces pristinaespiralis

A terpene cyclase from Streptomyces pristinaespiralis was characterized as the synthase for (+)‐(2S,3S,9R)‐pristinol. The structure of this sesquiterpene alcohol, which has a new carbon skeleton, was established by NMR spectroscopy and single‐wavelength anomalous‐dispersion X‐ray crystallography. Extensive isotopic labelling experiments were performed to distinguish between various possible cyclization mechanisms of the terpene cyclase and to decipher the EI‐MS fragmentation mechanism for pristinol.     

Isopentenyl diphosphate isomerase. Mechanism-based inhibition by diene analogues of isopentenyl diphosphate and dimethylallyl diphosphate

Isopentenyl diphosphate isomerase (IDI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). This is an essential step in the mevalonate entry into the isoprenoid biosynthetic pathway. The isomerization catalyzed by type I IDI involves protonation of the carbon-carbon double bond in IPP or DMAPP to form a tertiary carbocation, followed by deprotonation. Diene analogues for DMAPP (E-2-OPP and Z-2-OPP) and IPP (4-OPP) were synthesized and found to be potent active-site-directed irreversible inhibitors of the enzyme. X-ray analysis of the E.I complex between Escherichia coli IDI and 4-OPP reveals the presence of two isomers that differ in the stereochemistry of the newly formed C3-C4 double bond in the hydrocarbon chain of the inhibitor. In both adducts C5 of the inhibitor is joined to the sulfur of C67. In these structures the methyl group formed upon protonation of the diene moiety in 4-OPP is located near E116, implicating that residue in the protonation step.     

Synthesis of farnesyl diphosphate analogues containing ether-linked photoactive benzophenones and their application in studies of protein prenyltransferases

Protein prenylation is a posttranslational lipid modification in which C(15) and C(20) isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.     

Structures and Biosynthesis of Corvol Ethers—Sesquiterpenes from the Actinomycete Kitasatospora setae

Here we present the functional characterization of a sesquiterpene cyclase from Kitasatospora setae. The enzyme converts the sesquiterpene precursor farnesyl diphosphate (FPP) into two previously unknown and unstable sesquiterpene ethers for which we propose the trivial names corvol ethers A and B. Both compounds were purified and their structures were determined by one‐ and two‐dimensional NMR spectroscopy. A biosynthetic mechanism for the FPP cyclization by the corvol ether synthase was proposed. The results from the incubation experiments of the corvol ether synthase with isotopically labeled precursors were in line with this mechanism, while alternative mechanisms could clearly be ruled out.     

Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases

Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His?-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-2H?]FPP (1a), (1S)-[1-2H]-FPP (1b), and (1R)-[1-2H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.     

Stereochemistry of the macrocyclization and elimination steps in taxadiene biosynthesis through deuterium labeling

Taxadiene synthase catalyzes the cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) to taxa-4(5),11(12)-diene (Scheme 1, 5 --> 2) as the first committed step of Taxol biosynthesis. Deuterated GGPPs labeled stereospecifically at C-1, C-4, and C-16 were synthesized and incubated with recombinant taxadiene synthase from Taxus brevifolia to elucidate the stereochemistry of the cyclization reaction at these positions. The deuterium-labeled taxadienes obtained from (R)-[1-(2H1)]-, (S)-[1-(2H1)]-, and [16,16,16-(2H3)]GGPPs (9, 10, and 23b) were established to have deuterium in the 2alpha and 2beta CH2 and 16CH3 positions, respectively, by high-field 1H NMR spectroscopy (eqs 1-3). Incubation of (R)-[4-(2H1)]GGPP (17) with the recombinant enzyme gave a 10:10:80 mixture of [5beta-(2H1)]taxa-3(4),11(12)-diene, [5beta-(2H1)]taxa-4(20),11(12)-diene, and unlabeled taxa-4(5),11(12)-diene according to GC/MS analyses of the products (eq 4). It follows that C-1 of GGPP underwent inversion of configuration, that the A ring cyclization occurs on the si face of C15, and that the terminating proton abstraction removes H5beta from the final taxenyl carbocation intermediate. Thus, the C1-C14 and C15-C10 bonds are formed on the opposite faces of the 14,15 double bond of the substrate, i.e., overall anti electrophilic addition. The implications of these findings for the mechanism of the cyclization and rearrangement are discussed.     

Mechanism and stereochemistry of the germacradienol/germacrene D synthase of Streptomyces coelicolor A3(2)

Incubation of farnesyl diphosphate (1, FPP) with recombinant germacradienol synthase from Streptomyces coelicolor A3(2) gave, in addition to (4S,7R)-germacra-1(10)E,5E-diene-11-ol (2), 15% of (-)-germacrene D (5). Incubations of [1,1-2H2]FPP (1a), (1R)-[1-2H]FPP (1b), and (1S)-[1-2H]FPP (1c) with germacradienol/germacrene D synthase and analysis of the resulting samples of germacradienol (2) and germacrene D (5) by a combination of 1H, 2H, and 13C NMR and mass spectrometry established that it is H-1si of FPP that is lost in the formation of germacradienol (2) and that undergoes 1,3-hydride transfer in the formation of (-)-germacrene D (5). The proportion of the two products was also sensitive to isotopic labeling, with cyclization of (1S)-[1-2H]FPP (1c) giving an increased proportion (35%) of 5. These results could be explained by a mechanism involving partitioning of a common helminthogermacradienyl cation intermediate 7.     

Cytochrome P450 Mediated Cyclization in Eunicellane Derived Diterpenoid Biosynthesis**

Terpene cyclization, one of the most complex chemical reactions in nature, is generally catalyzed by two classes of terpene cyclases (TCs). Cytochrome P450s that act as unexpected TC-like enzymes are known but are very rare. In this study, we genome-mined a cryptic bacterial terpenoid gene cluster, named ari, from the thermophilic actinomycete strain Amycolatopsis arida. By employing a heterologous production system, we isolated and characterized three highly oxidized eunicellane derived diterpenoids, aridacins A−C ( 1 – 3 ), that possess a 6/7/5-fused tricyclic scaffold. In vivo and in vitro experiments systematically established a noncanonical two-step biosynthetic pathway for diterpene skeleton formation. First, a class I TC (AriE) cyclizes geranylgeranyl diphosphate (GGPP) into a 6/10-fused bicyclic cis-eunicellane skeleton. Next, a cytochrome P450 (AriF) catalyzes cyclization of the eunicellane skeleton into the 6/7/5-fused tricyclic scaffold through C2−C6 bond formation. Based on the results of quantum chemical computations, hydrogen abstraction followed by electron transfer coupled to barrierless carbocation ring closure is shown to be a viable mechanism for AriF-mediated cyclization. The biosynthetic logic of skeleton construction in the aridacins is unprecedented, expanding the catalytic capacity and diversity of P450s and setting the stage to investigate the inherent principles of carbocation generation by P450s in the biosynthesis of terpenoids.     

Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method,and Elucidation of its Biosynthesis

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene ( 1 ), having a 6‐8‐6‐5‐fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope‐incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation‐initiated second cyclization sequence, 1,2‐alkyl migration, and 1,5‐hydride shift to generate the novel scaffold of 1 .     

Astellifadiene: Structure Determination by NMR Spectroscopy and Crystalline Sponge Method,and Elucidation of its Biosynthesis

Genome mining of a terpene synthase gene from Emericella variecolor NBRC 32302 and its functional expression in Aspergillus oryzae led to the production of the new sesterterpene hydrocarbon, astellifadiene ( 1 ), having a 6‐8‐6‐5‐fused ring system. The structure of 1 was initially investigated by extensive NMR analyses, and was further confirmed by the crystalline sponge method, which established the absolute structure of 1 and demonstrated the usefulness of the method in the structure determination of complex hydrocarbon natural products. Furthermore, the biosynthesis of 1 was proposed on the basis of isotope‐incorporation experiments performed both in vivo and in vitro. The cyclization of GFPP involves a protonation‐initiated second cyclization sequence, 1,2‐alkyl migration, and 1,5‐hydride shift to generate the novel scaffold of 1 .     

Orchestration of discoid polyketide cyclization in the resistomycin pathway

Resistomycin is a bacterial polyphenolic metabolite from Streptomyces resistomycificus with a unique pentacyclic "discoid" ring system that clearly differs from the typical linear or angular architectures of aromatic polyketides. The first comprehensive cyclase amino acid sequence-function correlation revealed that the enzymes directing the nascent polyketide chain into a peri-fused system clearly differ from canonical linear and angular cyclases. All genes that are required and sufficent for resistomycin (rem) biosynthesis were identified through systematic dissection and reconstitution of the type II polyketide synthase (PKS) complex. The minimal rem PKS and the first cyclase were successfully cross-complemented with orthologues from the linear tetracenomycin polyketide pathway, indicating that both dekaketide pathways share early biosynthetic steps. In total three cyclases that are involved in discoid cyclization (RemI, RemF, and RemL) were identified by mutational analyses and in vivo pathway reconstitution. Analyses of the metabolic profiles of mutants expressing incomplete gene sets led to the discovery of a novel tetracenomycin derivative, TcmR1. The most surprising finding is that only the concerted action of the PKS and all three cyclases leads to the discoid ring structure. These results provide strong support for a model according to which the multienzyme complex forms a cage in which the polyketide is shaped, rather than a sequential cyclization of the polyketide chain by individual enzymes.     

Heterologous expression, purification, reconstitution and kinetic analysis of an extended type II polyketide synthase.

BACKGROUND: Polyketide synthases (PKSs) are bacterial multienzyme systems that synthesize a broad range of natural products. The 'minimal' PKS consists of a ketosynthase, a chain length factor, an acyl carrier protein and a malonyl transferase. Auxiliary components (ketoreductases, aromatases and cyclases are involved in controlling the oxidation level and cyclization of the nascent polyketide chain. We describe the heterologous expression and reconstitution of several auxiliary PKS components including the actinorhodin ketoreductase (act KR), the griseusin aromatase/cyclase (gris ARO/CYC), and the tetracenomycin aromatase/cyclase (tcm ARO/CYC). RESULTS: The polyketide products of reconstituted act and tcm PKSs were identical to those identified in previous in vivo studies. Although stable protein-protein interactions were not detected between minimal and auxiliary PKS components, kinetic analysis revealed that the extended PKS comprised of the act minimal PKS, the act KR and the gris ARO/CYC had a higher turnover number than the act minimal PKS plus the act KR or the act minimal PKS alone. Adding the tcm ARO/CYC to the tcm minimal PKS also increased the overall rate. CONCLUSIONS: Until recently the principal strategy for functional analysis of PKS subunits was through heterologous expression of recombinant PKSs in Streptomyces. Our results corroborate the implicit assumption that the product isolated from whole-cell systems is the dominant product of the PKS. They also suggest that an intermediate is channeled between the various subunits, and pave the way for more detailed structural and mechanistic analysis of these multienzyme systems.