Abstract
Most carotenoids are C40 metabolites produced from C20 geranylgeranyl diphosphate (GGPP). The enzymes that produce this precursor, GGPP synthases (GGPPS), are members of the short-chain prenyltransferase (SC-PT) family. SC-PTs are enzymes that catalyze the sequential head-to-tail addition of one or more C5 molecules of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) with the concomitant release of pyrophosphate (PPi). SC-PTs produce linear isoprenyl diphosphates of up to C20 (GGPP) that serve as precursors for many groups of isoprenoids with a wide range of essential biological functions in Eucarya, Bacteria, and Archaea. Enzymatic analysis of SC-PT activity normally requires complex, laborious and expensive methods such as radioactivity-based assays or liquid chromatography–mass spectrometry (LC-MS). Here we describe a fast and inexpensive spectrophotometric protocol for determining the kinetic parameters of SC-PTs in purified enzyme preparations, using an adapted assay for PPi quantification. We developed the method using the Arabidopsis thaliana GGPPS11 enzyme, which produces geranylgeranyl diphosphate for the synthesis of carotenoids in the chloroplast.
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References
Rodríguez-Concepción M, Boronat A (2015) Breaking new ground in the regulation of the early steps of plant isoprenoid biosynthesis. Curr Opin Plant Biol 25:17–22. https://doi.org/10.1016/j.pbi.2015.04.001
Liang PH, Ko TP, Wang AHJ (2002) Structure, mechanism and function of prenyltransferases. Eur J Biochem 269:3339–3354. https://doi.org/10.1046/j.1432-1033.2002.03014.x
Vandermoten S, Haubruge É, Cusson M (2009) New insights into short-chain prenyltransferases: structural features, evolutionary history and potential for selective inhibition. Cell Mol Life Sci 66:3685–3695. https://doi.org/10.1007/s00018-009-0100-9
Ambo T, Noike M, Kurokawa H, Koyama T (2008) Cloning and functional analysis of novel short-chain cis-prenyltransferases. Biochem Biophys Res Commun 375:536–540. https://doi.org/10.1016/j.bbrc.2008.08.057
Sallaud C, Rontein D, Onillon S, Jabes F, Duffe P, Giacalone C, Thoraval S, Escoffier C, Herbette G, Leonhardt N, Causse M, Tissier A (2009) A novel pathway for sesquiterpene biosynthesis from Z,Z-farnesyl pyrophosphate in the wild tomato Solanum habrochaites. Plant Cell 21:301–317. https://doi.org/10.1105/tpc.107.057885
Schilmiller AL, Schauvinhold I, Larson M, Xu R, Charbonneau AL, Schmidt A, Wilkerson C, Last RL, Pichersky E (2009) Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proc Natl Acad Sci 106:10865–10870. https://doi.org/10.1073/pnas.0904113106
Hsieh F-L, Chang T-H, Ko T-P, Wang AH-J (2011) Structure and mechanism of an Arabidopsis medium/long-chain-length prenyl pyrophosphate synthase. Plant Physiol 155:1079–1090. https://doi.org/10.1104/pp.110.168799
Akhtar TA, Matsuba Y, Schauvinhold I, Yu G, Lees HA, Klein SE, Pichersky E (2013) The tomato cis-prenyltransferase gene family. Plant J 73:640–652. https://doi.org/10.1111/tpj.12063
Marrero PF, Poulter CD, Edwards PA (1992) Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity. J Biol Chem 267:21873–21878
Joly A, Edwards PA (1993) Effect of site-directed mutagenesis of conserved aspartate and arginine residues upon farnesyl diphosphate synthase activity. J Biol Chem 268:26983–26989
Tarshis LC, Yan M, Poulter CD, Sacchettini JC (1994) Crystal structure of recombinant farnesyl diphosphate synthase at 2.6-ANG. resolution. Biochemistry 33:10871–10877. https://doi.org/10.1021/bi00202a004
Song L, Poulter CD (1994) Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II. Proc Natl Acad Sci U S A 91:3044–3048
Koyama T, Tajima M, Sano H, Doi T, Koike-Takeshita A, Obata S, Nishino T, Ogura K (1996) Identification of significant residues in the substrate binding site of Bacillus stearothermophilus farnesyl diphosphate synthase. Biochemistry 35:9533–9538. https://doi.org/10.1021/bi960137v
Koyama T, Gotoh Y, Nishino T (2000) Intersubunit location of the active site of farnesyl diphosphate synthase: reconstruction of active enzymes by hybrid-type heteromeric dimers of site-directed mutants. Biochemistry 39:463–469
Aaron JA, Christianson DW (2010) Trinuclear metal clusters in catalysis by terpenoid synthases. Pure Appl Chem 82:1585–1597. https://doi.org/10.1351/PAC-CON-09-09-37
Ohnuma S, Hirooka K, Hemmi H, Ishida C, Ohto C, Nishino T (1996) Conversion of product specificity of archaebacterial geranylgeranyl-diphosphate synthase. J Biol Chem 271:18831–18837. https://doi.org/10.1074/jbc.271.31.18831
Ohnuma SI, Narita K, Nakazawa T, Ishida C, Takeuchi Y, Ohto C, Nishino T (1996) A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product. J Biol Chem 271:30748–30754
Tarshis LC, Proteau PJ, Kellogg BA, Sacchettini JC, Poulter CD (1996) Regulation of product chain length by isoprenyl diphosphate synthases. Proc Natl Acad Sci 93:15018–15023. https://doi.org/10.1073/pnas.93.26.15018
Wang K, Ohnuma S (1999) Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution. Trends Biochem Sci 24:445–451
Stanley Fernandez SM, Kellogg BA, Poulter CD (2000) Farnesyl diphosphate synthase. Altering the catalytic site to select for geranyl diphosphate activity. Biochemistry 39:15316–15321
Nagel R, Bernholz C, Vranová E, Košuth J, Bergau N, Ludwig S, Wessjohann L, Gershenzon J, Tissier A, Schmidt A (2015) Arabidopsis thaliana isoprenyl diphosphate synthases produce the C 25 intermediate, geranylfarnesyl diphosphate. Plant J 84:847–859. https://doi.org/10.1111/tpj.13064
Wang C, Chen Q, Fan D, Li J, Wang G, Zhang P (2016) Structural analyses of short-chain prenyltransferases identify an evolutionarily conserved GFPPS clade in Brassicaceae plants. Mol Plant 9:195–204. https://doi.org/10.1016/j.molp.2015.10.010
Cunillera N, Arró M, Delourme D, Karst F, Boronat A, Ferrer A (1996) Arabidopsis thaliana contains two differentially expressed farnesyl-diphosphate synthase genes. J Biol Chem 271:7774–7780. https://doi.org/10.1074/jbc.271.13.7774
Masferrer A, Arró M, Manzano D, Schaller H, Fernández-Busquets X, Moncaleán P, Fernández B, Cunillera N, Boronat A, Ferrer A (2002) Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase (FPS1S) in transgenic Arabidopsis induces a cell death/senescence-like response and reduced cytokinin levels. Plant J 30:123–132
Chang T-H, Hsieh F-L, Ko T-P, Teng K-H, Liang P-H, Wang AH-J (2010) Structure of a heterotetrameric geranyl pyrophosphate synthase from mint (Mentha piperita) reveals Intersubunit regulation. Plant Cell 22:454–467. https://doi.org/10.1105/tpc.109.071738
Kim OT, Bang KH, Jung SJ, Kim YC, Hyun DY, Kim SH, Cha SW (2010) Molecular characterization of ginseng farnesyl diphosphate synthase gene and its up-regulation by methyl jasmonate. Biol Plant 54:47–53. https://doi.org/10.1007/s10535-010-0007-1
Schmidt A, Wächtler B, Temp U, Krekling T, Séguin A, Gershenzon J (2010) A bifunctional geranyl and geranylgeranyl diphosphate synthase is involved in terpene oleoresin formation in Picea abies. Plant Physiol 152:639–655. https://doi.org/10.1104/pp.109.144691
Arró M, Manzano D, Ferrer A (2014) Farnesyl diphosphate synthase assay. Methods Mol Biol 1153:41–53. https://doi.org/10.1007/978-1-4939-0606-2_4
Henneman L, van Cruchten AG, Denis SW, Amolins MW, Placzek AT, Gibbs RA, Kulik W, Waterham HR (2008) Detection of nonsterol isoprenoids by HPLC-MS/MS. Anal Biochem 383:18–24. https://doi.org/10.1016/j.ab.2008.08.023
Nagel R, Gershenzon J, Schmidt A (2012) Nonradioactive assay for detecting isoprenyl diphosphate synthase activity in crude plant extracts using liquid chromatography coupled with tandem mass spectrometry. Anal Biochem 422:433–438. https://doi.org/10.1016/j.ab.2011.12.037
Ruiz-Sola MÁ, Barja MV, Manzano D, Llorente B, Schipper B, Beekwilder J, Rodriguez-Concepcion M (2016) A single gene encodes two differentially targeted geranylgeranyl diphosphate synthase isoforms. Plant Physiol 172:1393–1402. https://doi.org/10.1104/pp.16.01392
Ruiz-Sola MÁ, Coman D, Beck G, Barja MV, Colinas M, Graf A, Welsch R, Rütimann P, Bühlmann P, Bigler L, Gruissem W, Rodríguez-Concepción M, Vranová E (2016) Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. New Phytol 209:252–264. https://doi.org/10.1111/nph.13580
Wang G, Dixon R (2009) A heterodimeric geranyl(geranyl)diphosphate synthase from hop (Humulus lupulus) and the evolution of monoterpene biosynthesis. Proc Natl Acad Sci U S A 106:9914–9919. https://doi.org/10.1073/pnas.0904069106
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Barja, M.V., Rodríguez-Concepción, M. (2020). A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases. In: Rodríguez-Concepción, M., Welsch, R. (eds) Plant and Food Carotenoids. Methods in Molecular Biology, vol 2083. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9952-1_2
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DOI: https://doi.org/10.1007/978-1-4939-9952-1_2
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