- Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity
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We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.
- Gerasymenko, Iryna,Sheludko, Yuriy V.,Navarro Fuertes, Ismael,Schmidts, Volker,Steinel, Lara,Haumann, Elisabeth,Warzecha, Heribert
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- Identification of a bacteria-produced benzisoxazole with antibiotic activity against multi-drug resistant Acinetobacter baumannii
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The emergence of multi-drug resistant pathogenic bacteria represents a serious and growing threat to national healthcare systems. Most pressing is an immediate need for the development of novel antibacterial agents to treat Gram-negative multi-drug resistant infections, including the opportunistic, hospital-derived pathogen, Acinetobacter baumannii. Herein we report a naturally occurring 1,2-benzisoxazole with minimum inhibitory concentrations as low as 6.25 μg ml?1 against clinical strains of multi-drug resistant A. baumannii and investigate its possible mechanisms of action. This molecule represents a new chemotype for antibacterial agents against A. baumannii and is easily accessed in two steps via de novo synthesis. In vitro testing of structural analogs suggest that the natural compound may already be optimized for activity against this pathogen. Our results demonstrate that supplementation of 4-hydroxybenzoate in minimal media was able to reverse 1,2-benzisoxazole’s antibacterial effects in A. baumannii. A search of metabolic pathways involving 4-hydroxybenzoate coupled with molecular modeling studies implicates two enzymes, chorismate pyruvate-lyase and 4-hydroxybenzoate octaprenyltransferase, as promising leads for the target of 3,6-dihydroxy-1,2-benzisoxazole.
- Deering, Robert W.,Whalen, Kristen E.,Alvarez, Ivan,Daffinee, Kathryn,Beganovic, Maya,LaPlante, Kerry L.,Kishore, Shreya,Zhao, Sijing,Cezairliyan, Brent,Yu, Shen,Rosario, Margaret,Mincer, Tracy J.,Rowley, David C.
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p. 370 - 380
(2021/02/22)
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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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supporting information
p. 8486 - 8490
(2020/03/30)
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- Algal neurotoxin biosynthesis repurposes the terpene cyclase structural fold into an N-prenyltransferase
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Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family.
- Chekan, Jonathan R.,McKinnie, Shaun M. K.,Moore, Bradley S.,Noel, Joseph P.
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p. 12799 - 12805
(2020/06/17)
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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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p. 7461 - 7469
(2018/10/24)
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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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- Structural Characterization of Early Michaelis Complexes in the Reaction Catalyzed by (+)-Limonene Synthase from Citrus sinensis Using Fluorinated Substrate Analogues
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The stereochemical course of monoterpene synthase reactions is thought to be determined early in the reaction sequence by selective binding of distinct conformations of the geranyl diphosphate (GPP) substrate. We explore here formation of early Michaelis complexes of the (+)-limonene synthase [(+)-LS] from Citrus sinensis using monofluorinated substrate analogues 2-fluoro-GPP (FGPP) and 2-fluoroneryl diphosphate (FNPP). Both are competitive inhibitors for (+)-LS with KI values of 2.4 ± 0.5 and 39.5 ± 5.2 μM, respectively. The KI values are similar to the KM for the respective nonfluorinated substrates, indicating that fluorine does not significantly perturb binding of the ligand to the enzyme. FGPP and FNPP are also substrates, but with dramatically reduced rates (kcat values of 0.00054 ± 0.00005 and 0.00024 ± 0.00002 s-1, respectively). These data are consistent with a stepwise mechanism for (+)-LS involving ionization of the allylic GPP substrate to generate a resonance-stabilized carbenium ion in the rate-limiting step. Crystals of apo-(+)-LS were soaked with FGPP and FNPP to obtain X-ray structures at 2.4 and 2.2 ? resolution, respectively. The fluorinated analogues are found anchored in the active site through extensive interactions involving the diphosphate, three metal ions, and three active-site Asp residues. Electron density for the carbon chains extends deep into a hydrophobic pocket, while the enzyme remains mostly in the open conformation observed for the apoprotein. While FNPP was found in multiple conformations, FGPP, importantly, was in a single, relatively well-defined, left-handed screw conformation, consistent with predictions for the mechanism of stereoselectivity in the monoterpene synthases.
- Kumar, Ramasamy P.,Morehouse, Benjamin R.,Matos, Jason O.,Malik, Karan,Lin, Hongkun,Krauss, Isaac J.,Oprian, Daniel D.
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p. 1716 - 1725
(2017/04/04)
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- A detailed view on 1,8-cineol biosynthesis by Streptomyces clavuligerus
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The stereochemical course of the cyclisation reaction catalysed by the bacterial 1,8-cineol synthase from Streptomyces clavuligerus was investigated using stereospecifically deuterated substrates. In contrast to the well investigated plant enzyme from Salvia officinalis, the reaction proceeds via (S)-linalyl diphosphate and the (S)-terpinyl cation, while the final cyclisation reaction is in both cases a syn addition, as could be shown by incubation of (2-13C)geranyl diphosphate in deuterium oxide.
- Rinkel, Jan,Rabe, Patrick,Zur Horst, Laura,Dickschat, Jeroen S.
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supporting information
p. 2317 - 2324
(2016/11/17)
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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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- Biosynthesis of monoterpene scent compounds in roses
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The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta.
- Magnard, Jean-Louis,Roccia, Aymeric,Caissard, Jean-Claude,Vergne, Philippe,Sun, Pulu,Hecquet, Romain,Dubois, Annick,Oyant, Laurence Hibrand-Saint,Jullien, Frédéric,Nicolè, Florence,Raymond, Olivier,Huguet, Stéphanie,Baltenweck, Raymonde,Meyer, Sophie,Claudel, Patricia,Jeauffre, Julien,Rohmer, Michel,Foucher, Fabrice,Hugueney, Philippe,Bendahmane, Mohammed,Baudino, Sylvie
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- Biosynthesis of ambiguine indole alkaloids in cyanobacterium Fischerella ambigua
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Ambiguines belong to a family of hapalindole-type indole alkaloid natural products, with many of the members possessing up to eight consecutive carbon stereocenters in a fused pentacyclic 6-6-6-5-7 ring scaffold. Here, we report the identification of a 42 kbp ambiguine (amb) biosynthetic gene cluster that harbors 32 protein-coding genes in its native producer Fischerella ambigua UTEX1903. Association of the amb cluster with ambiguine biosynthesis was confirmed by both bioinformatic analysis and in vitro characterizations of enzymes responsible for 3-((Z)-2′-isocyanoethenyl) indole and geranyl pyrophosphate biosynthesis and a C-2 indole dimethylallyltransferase that regiospecifically tailors hapalindole G to ambiguine A. The presence of five nonheme iron-dependent oxygenase coding genes (including four Rieske-type oxygenases) within the amb cluster suggests late-stage C-H activations are likely responsible for the structural diversities of ambiguines by regio- and stereospecific chlorination, hydroxylation, epoxidation, and sp 2-sp3 C-C bond formation.
- Hillwig, Matthew L.,Zhu, Qin,Liu, Xinyu
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p. 372 - 377
(2014/03/21)
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- Characterization of thermophilic archaeal isopentenyl phosphate kinases
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Archaea synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), the essential building blocks of isoprenoid compounds, from mevalonate (MVA). However, an analysis of the genomes of several members of the Archaea failed to identify genes for the enzymes required to convert phosphomevalonate (PM) to IPP in eukaryotes. The recent discovery of an isopentenyl kinase (IPK) in Methanocaldococcus jannaschii (MJ) suggests a new variation of the MVA pathway where PM is decarboxylated to give isopentenyl phosphate (IP), which is phosphorylated to produce IPP. A blast search using the MJ protein as a probe revealed a subfamily of amino acid kinases that include the fosfomycin resistance protein fomA, which deactivates the antibiotic by phosphorylation of its phosphonate residue in a reaction similar to the conversion of IP to IPP. IPK genes were cloned from two organisms identified in the search, Methanothermobacter thermautotrophicus (MTH) and Thermoplasma acidophilum (THA), and the His-tagged recombinant proteins were purified by Ni-NTA chromatography. The enzymes catalyze the reversible phosphorylation of IP by ATP, Keq=6.3 ± 1. The catalytic efficiencies (V/K) of the proteins were ～2 × 106M-1 s-1. In the reverse direction, ADP was a substrate inhibitor for THAIPK, Ki ADP=58 ±6 μM, but not forMTHIPK. Both enzymes were active over a broad range of pH and temperature. Five compounds, dimethylallyl phosphate, isopentenyl thiolophosphate, 1-butyl phosphate, 3-buten-1-yl phosphate, and geranyl phosphate, were evaluated as alternative substrates for the MTH and THA IP kinases. All of the compounds were phosphorylated, although the catalytic efficiency was low for geranyl phosphate. 2009 American Chemical Society.
- Chen, Mo,Poulter, C. Dale
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experimental part
p. 207 - 217
(2010/10/21)
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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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p. 2649 - 2659
(2008/09/18)
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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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- 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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p. 1016 - 1018
(2007/10/03)
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- Natural and unnatural terpenoid precursors of insect juvenile hormone
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The biosynthesis of insect juvenile hormone (JH) is due, in part, to the precise head-to-tail coupling of allylic and homoallylic diphosphate substrates, as catalyzed by one or more prenyltransferases. To better understand this enzyme's role in JH production, homodimethylallyl diphosphate and both the natural and unnatural homologs of geranyl diphosphate have been prepared as potential substrates for insect prenyltransferase. These latter materials were constructed in a convergent manner by olefination of the corresponding trisnoraldehydes obtained from either terminal oxidative cleavage of geraniol or higher-order cuprate conjugate addition to acrolein. To aid in characterizing the nature of the terpenoid skeletons formed from our in vitro studies, homologous derivatives of farnesol were also prepared by anion coupling of the geranyl derivatives to either C5 or C6 allylic bromides. The preparation of these materials and the results of incubations with larval corpora allata homogenates of the lepidopteran Manduca sexta are described.
- Sen, Stephanie E.,Ewing, Gregory J.
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p. 3529 - 3536
(2007/10/03)
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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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- 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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p. 1967 - 1969
(2007/10/02)
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