- THE ABSOLUTE STEREOCHEMISTRY OF THE ENZYMIC CYCLISATION TO FORM THE STERPURENE SESQUITERPENES
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Incorporation studies using acetate into a novel sterpurene sesquiterpene, 9,12-dihydroxysterpurene (1), have allowed the absolute stereochemistry of the enzymic cyclisation of farnesyl pyrophosphate to sterpurene to be elucidated.Observation of two-bond 13C-13C coupling across the cyclobutane ring confirms the derivation of these two carbon atoms from the same acetate unit.
- Abell, Chris,Leech, Andrew P.
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- Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase.
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[formula: see text] Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes.
- Leyes,Baker,Poulter
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- Mycobacterium tuberculosis H37Rv3377c encodes the diterpene cyclase for producing the halimane skeleton
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The cloning and functional expression of Mycobacterium tuberculosis Rv3377c in Escherichia coli revealed that this gene encodes the diterpene cyclase for producing (+)-5(6),13-halimadiene-15-ol, which accepts geranylgeranyldiphosphate as the intrinsic substrate. The Royal Society of Chemistry 2005.
- Nakano, Chiaki,Okamura, Tomoo,Sato, Tsutomu,Dairi, Tohru,Hoshino, Tsutomu
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- Incorporation of farnesyl pyrophosphate derivertives into abscisic acid and its biosynthetic intermediates in Cercospora cruenta
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To investigate the transformation from (2E,6E)-farnesyl pyrophosphate to (2Z,4E)-γ-ionylideneethanol in the abscisic acid-producing fungi, Cercospora cruenta, plausible [2-14C]-C15 intermediates were prepared and fed. Substrates such as (2E,6E)-farnesyl pyrophosphate, (2Z,4E)-γ-ionylideneethanol and its pyrophosphate were incorporated into ABA and its known biosynthetic precursors. It is suggested that (2E,6E)-farnesyl pyrophosphate is converted to (2Z,4E)-γ-ionylideneethanol in four consecutive steps: dehydrogenation, isomerization, cyclization and hydrolysis.
- Yamamoto, Hirotaka,Oritani, Takayuki
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- Cloning and characterization of isoprenyl diphosphate synthases with farnesyl diphosphate and geranylgeranyl diphosphate synthase activity from Norway spruce (Picea abies) and their relation to induced oleoresin formation
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The conifer Picea abies (Norway spruce) employs terpenoid-based oleoresins as part of its constitutive and induced defense responses to herbivores and pathogens. The isoprenyl diphosphate synthases are branch-point enzymes of terpenoid biosynthesis leading to the various terpene classes. We isolated three genes encoding isoprenyl diphosphate synthases from P. abies cDNA libraries prepared from the bark and wood of methyl jasmonate-treated saplings and screened via a homology-based PCR approach using degenerate primers. Enzyme assays of the purified recombinant proteins expressed in Escherichia coli demonstrated that one gene (PaIDS 4) encodes a farnesyl diphosphate synthase and the other two (PaIDS 5 and PaIDS 6) encode geranylgeranyl diphosphate synthases. The sequences have moderate similarity to those of farnesyl diphosphate and geranylgeranyl diphosphate synthases already known from plants, and the kinetic properties of the enzymes are not unlike those of other isoprenyl diphosphate synthases. Of the three genes, only PaIDS 5 displayed a significant increase in transcript level in response to methyl jasmonate spraying, suggesting its involvement in induced oleoresin biosynthesis.
- Schmidt, Axel,Gershenzon, Jonathan
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- Synthesis of geranyl S-thiolodiphosphate. A new alternative substrate/inhibitor for prenyltransferases.
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The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7) in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.
- Phan,Poulter
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- Tris(tetra-n-butylammonium) hydrogen pyrophosphate. A new reagent for the preparation of allylic pyrophosphate esters [3]
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Tris(tetra-n-butylammonium) hydrogen pyrophosphate was used to prepare dimethylallyl pyrophosphate (1-OPP), 7-methylocta-2,6-dien-1-yl pyrophosphate (2-OPP), geranyl pyrophosphate (3-OPP), 2-flourogeranyl pyrophosphate (4-OPP), and farnesyl pyrophosphate (5-OPP) from the corresponding alcohols in moderate yields by a two-step sequence via the corresponding primary, allylic bromides.
- Dixit, Vyas M.,Laskovics, F. Mark,Noall, Wendy I.,Poulter, C. Dale
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- Mutation of archaeal isopentenyl phosphate kinase highlights mechanism and guides phosphorylation of additional isoprenoid monophosphates
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The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg2+-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0-2.8 A. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε2 nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors.
- Dellas, Nikki,Noel, Joseph P.
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- Structure-based protein engineering enables prenyl donor switching of a fungal aromatic prenyltransferase
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Microorganisms provide valuable enzyme machinery to assemble complex molecules. Fungal prenyltransferases (PTs) typically catalyse highly regiospecific prenylation reactions that are of significant pharmaceutical interest. While the majority of PTs accepts dimethylallyl diphosphate (DMAPP), very few such enzymes can use geranyl diphosphate (GPP) or farnesyl diphosphate (FPP) as donors. This catalytic gap prohibits the wide application of PTs for structural diversification. Structure-guided molecular modelling and site-directed mutagenesis of FgaPT2 from Aspergillus fumigatus led to the identification of the gatekeeping residue Met328 responsible for the prenyl selectivity and sets the basis for creation of GPP- and FPP-accepting enzymes. Site-saturation mutagenesis of the gatekeeping residue at position 328 in FgaPT2 revealed that the size of this side chain is the determining factor for prenyl selectivity, while its hydrophobicity is crucial for allowing DMAPP and GPP to bind.
- Mai, Peter,Zocher, Georg,Stehle, Thilo,Li, Shu-Ming
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- Farnesyl Diphosphate Synthase Reactions of Geranyl Diphosphate Analogues Having Oxygen Atoms in Their Alkyl Chains
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Seven geranyl diphosphate analogues having oxygen atoms in their alkyl chains were synthesized and examined for their reactivities as substrates in the reaction catalyzed by pig liver farnesyl diphosphate synthase.All of these compounds acted as substrates to give farnesyl diphosphate analogues.It was suggested that the enzyme cavity for the geranyl moiety is tolerant enough to accommodate alkyl moietes containing oxygen atoms.
- Maki, Yuji,Satoh, Hikaru,Kurihara, Masayo,Endo, Takae,Watanabe, Gotaro,Ogura, Kyozo
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- Modular Chemoenzymatic Synthesis of Terpenes and their Analogues
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Non-natural terpenoids offer potential as pharmaceuticals and agrochemicals. However, their chemical syntheses are often long, complex, and not easily amenable to large-scale production. Herein, we report a modular chemoenzymatic approach to synthesize terpene analogues from diphosphorylated precursors produced in quantitative yields. Through the addition of prenyl transferases, farnesyl diphosphates, (2E,6E)-FDP and (2Z,6Z)-FDP, were isolated in greater than 80 % yields. The synthesis of 14,15-dimethyl-FDP, 12-methyl-FDP, 12-hydroxy-FDP, homo-FDP, and 15-methyl-FDP was also achieved. These modified diphosphates were used with terpene synthases to produce the unnatural sesquiterpenoid semiochemicals (S)-14,15-dimethylgermacrene D and (S)-12-methylgermacrene D as well as dihydroartemisinic aldehyde. This approach is applicable to the synthesis of many non-natural terpenoids, offering a scalable route free from repeated chain extensions and capricious chemical phosphorylation reactions.
- Allemann, Rudolf K.,Benton, Jennifer C. R.,Dunbabin, Alice,Johnson, Luke A.,Mart, Robert J.
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p. 8486 - 8490
(2020/03/30)
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- Structure-Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis
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The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A. tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A. tridentata FPPase and CPPase are located within a six-helix bundle containing amino acids 53 to 241. The two enzymes were metamorphosed into one another by sequentially replacing the loops and helices of the six-helix bundle from enzyme with those from the other. Chain elongation was the dominant activity during the N-terminal to C-terminal metamorphosis of FPPase to CPPase, with product selectivity gradually switching from FPP to GPP, until replacement of the final α-helix, whereupon cyclopropanation and branching activity competed with chain elongation. During the corresponding metamorphosis of CPPase to FPPase, cyclopropanation and branching activities were lost upon replacement of the first helix in the six-helix bundle. Mutations of active site residues in CPPase to the corresponding amino acids in FPPase enhanced chain-elongation activity, while similar mutations in the active site of FPPase failed to significantly promote formation of significant amounts of irregular monoterpenes. Our results indicate that CPPase, a promiscuous enzyme, is more plastic toward acquiring new activities, whereas FPPase is more resistant. Mutations of residues outside of the α terpene synthase fold are important for acquisition of FPPase activity for synthesis of CPP, LPP, and MPP.
- Lee, J. Scott,Pan, Jian-Jung,Ramamoorthy, Gurusankar,Poulter, C. Dale
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supporting information
p. 14556 - 14567
(2017/10/24)
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- Specificity of geranylgeranyl diphosphate synthase for homoallylic substrate analogs
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The goal of this study was to determine the substrate specificity of Homo sapiens geranylgeranyl diphosphate synthase (GGPPase) for analogs of isopentenyl diphosphate (IPP) to facilitate the application to organic synthesis techniques to the study of prenyl chain elongation enzymes. For this purpose, we used the IPP analogs 2a-d, which contain different alkyl side-chains at the 3-position, as substrates of the condensation reaction with the allylic substrate geranyl diphosphate (GPP) that is catalyzed by GGPPase. GGPPase catalyzed the reaction of GPP with 3-desmethylisopentenyl diphosphate (but-3-enyl diphosphate) to yield 3-desmethylfarnesyl diphosphate (12.1%), as well as the reaction of GPP with 3-ethylbut-3-enyl diphosphate or 3-propylbut-3-enyl diphosphate to yield 3-ethylfarnesyl diphosphate (46.9%) or 3-propylfarnesyl diphosphate (22.6%), respectively. However, a reaction product was not detected when 3-butylbut-3-enyl diphosphate was used as substrate.
- Ohya, Norimasa,Ichijo, Takumi,Sato, Hana,Nakamura, Takeshi,Yokota, Saki,Sagami, Hiroshi,Nagaki, Masahiko
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p. 179 - 182
(2015/09/01)
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- Isotope sensitive branching and kinetic isotope effects to analyse multiproduct terpenoid synthases from Zea mays
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Multiproduct terpene synthases TPS4-B73 and TPS5-Delprim from Zea mays exhibit isotopically sensitive branching in the formation of mono- and sesquiterpene volatiles. The impact of the kinetic isotope effects and the stabilization of the reactive intermediates by hyperconjugation along with the shift of products from alkenes to alcohols are discussed.
- Gatto, Nathalie,Vattekkatte, Abith,K?llner, Tobias,Degenhardt, J?rg,Gershenzon, Jonathan,Boland, Wilhelm
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supporting information
p. 3797 - 3800
(2015/03/30)
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- Substrate specificities of E- and Z-farnesyl diphosphate synthases with substrate analogs
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Prenyltransferases catalyzes the basic isoprenoid chain elongation to produce prenyl diphosphates, which led to upward of 30,000 diverse isoprenoids as steroids, carotenoids, natural rubbers, and prenyl proteins. Here, we determined the reactivities of E- and Z-farnesyl diphosphate synthases (E- and Z-FPP synthases) isolated from Bacillus stearothermophilus and Thermobifida fusca, respectively. For this purpose we use the synthetic substrate analogs, 8-tetrahydropyran-2-yloxy-, 8-hydroxy- and 8-acetoxygeranyl diphosphates. Z-FPP synthase catalyzed the reaction between 8-hydroxygeranyl diphosphate (HOGPP) and isopentenyl diphosphate (IPP), which produced (2Z)-12-hydroxyfarnesyl diphosphate (yield: 16.7%) and (2Z, 6Z)-16-hydroxygeranylgeranyl diphosphate (yield: 6.6%). Neither E- nor Z-farnesyl diphosphate synthases detectably catalyzed reactions between 8-tetrahydropyran-2-yloxygeranyl diphosphate (8-THPOGPP) and IPP. However, a mutated E-FPP synthase (Y81S), did catalyze this reaction, producing 12-tetrahydropyran-2-yloxyfarnesyl diphosphate (12-THPOFPP) with a yield of 12.3%. Wild-type E-FPP synthase catalyzed the reaction of 8-acetoxygeranyl diphosphate (8-AcOGPP) with IPP, which produced 12-acetoxyfarnesyl diphosphate (12-AcOFPP) (yield, 21.8%). Mutant E-FPP synthase catalyzed the reaction between 8-AcOGPP with IPP, producing 12-AcOFPP and 16-acetoxygeranylgeranyl diphosphate (16-AcOGGPP) with respective yields of 55.3% and 1.7%. We believe our results contribute to a better understanding of the catalytic properties of these key enzymes and illustrate their use in the stereo-specific syntheses of compounds that may have significant biotechnological and medical applications.
- Nagaki, Masahiko,Ichijo, Takumi,Kobashi, Rikiya,Yagihashi, Yusuke,Musashi, Tohru,Kawakami, Jun,Ohya, Norimasa,Gotoh, Takeshi,Sagami, Hiroshi
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experimental part
p. 1 - 6
(2012/09/05)
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- HIV-1 integrase inhibitor-inspired antibacterials targeting isoprenoid biosynthesis
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We report the discovery of antibacterial leads, keto- and diketo-acids, targeting two prenyl transferases: undecaprenyl diphosphate synthase (UPPS) and dehydrosqualene synthase (CrtM). The leads were suggested by the observation that keto- and diketo-acids bind to the active site Mg2+/Asp domain in HIV-1 integrase, and similar domains are present in prenyl transferases. We report the X-ray crystallographic structures of one diketo-acid and one keto-acid bound to CrtM, which supports the Mg2+ binding hypothesis, together with the X-ray structure of one diketo-acid bound to UPPS. In all cases, the inhibitors bind to a farnesyl diphosphate substrate-binding site. Compound 45 had cell growth inhibition MIC90 values of ～250-500 ng/mL against Staphylococcus aureus, 500 ng/mL against Bacillus anthracis, 4 μg/mL against Listeria monocytogenes and Enterococcus faecium, and 1 μg/mL against Streptococcus pyogenes M1 but very little activity against Escherichia coli (DH5α, K12) or human cell lines.
- Zhang, Yonghui,Lin, Fu-Yang,Li, Kai,Zhu, Wei,Liu, Yi-Liang,Cao, Rong,Pang, Ran,Lee, Eunhae,Axelson, Jordan,Hensler, Mary,Wang, Ke,Molohon, Katie J.,Wang, Yang,Mitchell, Douglas A.,Nizet, Victor,Oldfield, Eric
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p. 402 - 406
(2012/06/29)
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- Farnesyl diphosphate synthase: The art of compromise between substrate selectivity and stereoselectivity
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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.
- Thulasiram, Hirekodathakallu V.,Poulter, C. Dale
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p. 15819 - 15823
(2007/10/03)
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- Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors
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Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPPi) 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, KIGSPP=24.8 μM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.
- Phan,Poulter
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p. 6705 - 6710
(2007/10/03)
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- The Significance of the Diphosphate Linkage Involved in the Substrates for Prenyltransferase. Geranyl- and Dimethylallyl Methylenediphosphonate as Artificial Substrates
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Dimethylallyl- and geranyl methylenediphosphonate are active as artificial substrates for farnesyl diphosphate synthase to give E,E-farnesyl diphosphate.By comparing the kinetic data obtained with the analogues and the natural substrates, the significance of the diphosphate moiety of the substrates of prenyltransferase is discussed.
- Gotoh, Takeshi,Koyama, Tanetoshi,Ogura, Kyozo
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p. 1627 - 1630
(2007/10/02)
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- Phosphorylation of Isoprenoid Alcohols
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Procedures for the synthesis and purification of 20 isoprenoid diphosphates and methanediphosphonate analogues from the corresponding alcohols are described.The alcohols are activated for phosphorylation by conversion of homoallylic systems to tosylates and allylic systems to halides.The activated intermediates are treated with tris(tetra-n-butylammonium) salts of pyrophosphoric, methanediphosphonic, or difluoromethanediphosphonic acid to obtain the corresponding esters in yields 34-80percent.Chromatography on cellulose is a general method for purification of isoprenoid diphosphates, and procedures are decribed for compounds with C5 to C20 hydrocarbon moieties.The displacement by pyrophosphate occurs with inversion of configuration, and the procedure can be used to prepare isoprenoid diphosphates with chiral C1 methylene groups in high optical purity from the corresponding alcohols.
- Davisson, V. Jo,Woodside, Andrew B.,Neal, Timothy R.,Stremler, Kay E.,Muehlbacher, Manfred,Poulter, C. Dale
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p. 4768 - 4779
(2007/10/02)
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