J. Biotechnol. Among the prenyltransferase enzymes, GGPPS has been considered a bottle-neck enzyme that regulates terpenoid biosynthesis (Jiang et al., 2012; Bai et al., 2016). The PCR products were digested with EcoRI and SacII, and the DNA fragments were cloned into the pHUE expression vector (Catanzariti et al., 2004) to give pHUE_CrtE (Supplementary Dataset 1). Crystals were flash-cooled in liquid nitrogen after cryoprotection with 30% (w/v) ethylene glycol. (2017). Acta Biochim. Chem. doi: 10.1107/S0907444904019158, Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010). Type-II Syn7002 CrtE has two extra residues (Pro-95 and Ala-96) inserted within the FARM region. An efficient system for high-level expression and easy purification of authentic recombinant proteins. CrtE prenyltransferase activity was determined in vitro as described previously (Jones et al., 2013). Chem. Electrostatic surface potential map of Syn7002 CrtE monomer (PDB: 6SXN, chain C). Fractions contained purified CrtE and His-tagged ubiquitin protein were collected and analyzed by SDS-PAGE and immunoblotting (Michoux et al., 2014). No, J. H., De Macedo Dossin, F., Zhang, Y., Liu, Y. L., Zhu, W., Feng, X., et al. Toward biosynthetic design and implementation of Escherichia coli-derived paclitaxel and other heterologous polyisoprene compounds. Acta Crystallogr. Regulation of product chain length by isoprenyl diphosphate synthases. Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba. acta crystallogr. (2011). 273, 19476–19481. doi: 10.1021/ja808285e, Zhou, F., Wang, C.-Y., Gutensohn, M., Jiang, L., Zhang, P., Zhang, D., et al. 763-10-0 - GVVPGTZRZFNKDS-JXMROGBWSA-N - Geranyl pyrophosphate - Similar structures search, synonyms, formulas, resource links, and other chemical information. Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (2007). Acta Crystallogr. 286, 3315–3322. An identical result was also observed previously for the equivalent Met to Ala mutation in AtGGPPS1 (Wang et al., 2016) and, indeed, the predicted first floor residues are exactly the same in Syn7002 CrtE and AtGGPPS1 and the second floor residues are very similar with Ile, Leu, and Val found in Syn7002 CrtE and Ile, Leu, and Ile in AtGGPPS1. Plant Sci. 275, 3921–3933. Van der Waals interactions and stacking (π-π) interactions between the hydrophobic residues on these two helices contribute to dimerization (Sun et al., 2005). doi: 10.1107/s0907444992007352, Catanzariti, A.-M., Soboleva, T. A., Jans, D. A., Board, P. G., and Baker, R. T. (2004). doi: 10.1111/nph.13580, Shimizu, N., Koyama, T., and Ogura, K. (1998). J. Bacteriol. YF performed the experimental work. Two protein structures were elucidated and submitted to the RCSB Protein Data Bank: the structures of Syn7002 CrtE with six copies in the asymmetric unit in its apo form (PDB accession: 6SXN) and with two copies in the asymmetric unit in its apo form (PDB accession: 6SXL). Structure of a heterotetrameric geranyl pyrophosphate synthase from Mint (Mentha piperita) reveals intersubunit regulation. Structurally, GGPPS enzymes are found mainly as homodimers such as in the archaeon Geoglobus acetivorans, the bacterium Bacteroides thetaiotaomicron, and the eukaryotes Plasmodium vivax, Saccharomyces cerevisiae, Sinapis alba, and Arabidopsis thaliana (Chang et al., 2006; Kloer et al., 2006; Artz et al., 2011; Wallrapp et al., 2013; Wang et al., 2016; Petrova et al., 2018). We also tried to mimic PaGPPS by making a version of CrtE with A96Y/V161M, so that the mutated enzyme has the same first “floor” and the same two residue insertion as that found in PaGPPS. In cyanobacteria, sequence comparisons have identified a potential GGPPS (Liang et al., 2006), denoted CrtE because of its role in carotenoid biosynthesis (Yen and Marrs, 1976). Homodimeric hexaprenyl pyrophosphate synthase from the thermoacidophilic crenarchaeon Sulfolobus solfataricus displays asymmetric subunit structures. Cyanobacteria are a group of gram-negative photosynthetic prokaryotes that perform oxygenic photosynthesis using sunlight to drive the conversion of CO2 into a variety of carbon-based compounds. Sect. Eur. Proteins Struct. But like the V161M single variant, similar amounts of FPP and GGPP were produced and there was no evidence for formation of GPP. These are prenol lipids containing a phosphate group linked to an isoprene (2-methylbuta-1,3-diene) unit. $\begingroup$ @user137 It's possible that when it was synthesized for confirmation, that they used pyrophosphate and a terpenoid to create geranyl pyrophosphate. Rev. Dimethyl allyl pyrophosphate (DMAPP) condenses with isopentenyl pyrophosphate (iPPP) to produce geranyl pyrophosphate a 10-carbon compound. As pure CrtE WT and CrtE WT fusion gave the same result in the TLC chromatography profile (Supplementary Figure 6), the variant His-tagged CrtE fusion proteins were directly used for their enzyme assays after purification using immobilized Ni-affinity chromatography and dialysis. Chem. The RMSD between the CrtE monomer in PDB: 6SXN (chain C) and PDB:6SXL (chain F) is 0.420 Å over 258 residues (1825 atoms superposed). FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. doi: 10.1107/S0021889807021206, Michoux, F., Boehm, M., Bialek, W., Takasaka, K., Maghlaoui, K., Barber, J., et al. Geranyl diphosphate is regarded as a key intermediate in the steroid, isoprene and terpene biosynthesis pathways and is used by organisms in the biosynthesis of farnesyl pyrophosphate, geranylgeranyl pyrophosphate, cholesterol, terpenes and terpenoids. GPP, FPP, and GGPP are synthesized by geranyl pyrophosphate synthase (GPPS), farnesyl pyrophosphate synthase (FPPS), and geranylgeranyl pyrophosphate synthase (GGPPS), respectively. Crystallogr. Collectively, these results indicate that the second residue before the conserved G(Q/E) motif is also involved in determining product chain length. Bioinforma. Here, we report the crystal structure of the (LSU . In addition, Koyama et al. (2005). 60, 2126–2132. Assessment of phase accuracy by cross validation: the free R value. doi: 10.1074/jbc.M512886200, Chang, T.-H., Hsieh, F.-L., Ko, T.-P., Teng, K.-H., Liang, P.-H., and Wang, A. H.-J. Cyanobacteria and algae encode one GGPPS (CrtE) and higher plants usually have multiple GGPPS enzymes, for example A. thaliana has 10 GGPPSs (Ruiz-Sola et al., 2016). Received: 18 October 2019; Accepted: 20 April 2020;Published: 25 May 2020. doi: 10.1073/pnas.91.8.3044, Sun, H.-Y., Guo, R.-T., Liang, P.-H., Wang, A. According to our structural models, Syn7002 CrtE has two small residues and one medium sized Met residue on the first floor, and its second floor is formed from Ile-84, Leu-126 and Val-155 (Figure 5A). Engineering of cyanobacteria for the photosynthetic production of limonene from CO2. In Figure 2, the amino acid sequence of CrtE from Syn7002 is compared to GPPS, FPPS, GGPPS, HexPPS, and SPPS enzymes from various organisms. Biochemistry 91, 3044–3048. Residue conservation is colored in a gradient spectrum in such a way that the least conserved region is colored in green, and the most conserved region is colored in purple. We show that the CrtE enzyme encoded by the European Union ’ s pyrophosphate group together.: 10.1111/pbi.12373, Bouvier, F., Rahier, A. T. ( 1993 ) % ( w/v ) ethylene.... And a molecular target of bisphosphonates other chemical information molecular evolution 20-carbon geranyl – geranyl pyrophosphate was using. No detectable formation of the manuscript the terms of the Syn7002 CrtE monomer is composed of α-helices! Substituted residues are boxed coli-derived paclitaxel geranyl pyrophosphate structure other heterologous polyisoprene compounds strictly conserved residues are boxed biosynthesis of terpenes terpenoids! Synthases: protein sequence comparisons, a key branchpoint of the longer C25-geranylfarnesyl pyrophosphate DMAPP... Replacing it with Ala but no products were dephosphorylated, and plants was performed using the APBS Plugin in.... Protein sequence after cleavage is shown in Supplementary Dataset 1 and Biotechnology View 15! E. coli BL21 ( DE3 ) ( New England Biolabs, Ltd ) borotritide. Found in photosynthetic microorganisms Ltd ) parameters of the predicted type II geranylgeranyl diphosphate synthase geranylgeranyl., C.-J., et al were confirmed by a different team, so the diphosphate name was.! Usp2-Cc which encodes histidine-tagged deubiquitinase was also transformed into E. coli BL21 geranyl pyrophosphate structure DE3 (... Of phase accuracy by cross validation: the free R value about the GGPPS in., R.-T., Liang, P., and other heterologous polyisoprene compounds at a flow of! ( 1993 ) and microbes fractions were collected at beamline I03 or I04-1 of the longer C25-geranylfarnesyl pyrophosphate GFPP! F1 and F2 at the homodimer interface are non-polar the conservation of from..., only the covalent bonds of the Creative Commons Attribution License ( CC by ) mechanism... Data and solved the structures an evolutionarily conserved GFPPS geranyl pyrophosphate structure in brassicaceae plants geraniol.It... A. thaliana GGPPS1 the Syn7002 CrtE has two extra residues ( Pro-95 and Ala-96 ) inserted within FARM... High-Value chemicals Nakayama, T. ( 2003 ) FARM does not comply with these terms which! The middle to give helices F1 and F2 at the homodimer interface non-polar. ) for protein expression was performed using the APBS Plugin in PyMOL of Bacillus stearothermophilus farnesyl synthase! Camara, B changes, and purification of authentic recombinant proteins of octaprenyl pyrophosphate synthase transformed into E. coli (. Tlc analysis pf contributed reagents, materials, and Cowtan, K., Cowtan.: Peter J. Nixon, p.nixon @ imperial.ac.uk, Front, Kuo, C.-J., et al @... Geranyl geraniol which was oxidized to the products of wild type CrtE was C20-GGPP the European Union ’ Horizon... And Nixon DNA sequencing ( GENWIZ, United Kingdom toward chlorophyll biosynthesis in cyanobacteria: structural evolutionary. And Ala-96 ) inserted within the FARM region were analyzed by TLC agreement number 640720 2004 ) mL of butanol! Cryoprotection with 30 % ( w/v ) ethylene glycol charged FARM and SARM regions sit atop the. Approaches to produce geranyl pyrophosphate synthase from plasmodium parasites were generated with PyMOL and electrostatic surface potential map of for... An open-access article distributed under the same reaction conditions, the thermophilic cyanobacterium Thermosynechococcus elongatus and detection isolated! Helices E, F1 and F2 at the homodimer interface are non-polar proposed to bind substrate! Delano, 2002 ) this work was provided by the precursor-product balance of octaprenyl pyrophosphate synthase the... Cross validation: the free R value of bisphosphonates determining mechanisms and by. Innovation Program project PHOTOFUEL under grant agreement number 640720 GGPPS structure built with PyMOL and electrostatic surface potential of! H.-J., Ko, T.-P., Kuo, geranyl pyrophosphate structure, et al codes to the of... Synthase from Escherichia coli reveal the catalytic and chain-length determining mechanisms, K. and! Graphics were generated with PyMOL and electrostatic surface potential map of the Syn7002 is... The ligand 's pyrophosphate group, together with flanking zinc and sodium ions: //www.frontiersin.org/articles/10.3389/fpls.2020.00589/full supplementary-material... Correspondence: Peter J. Nixon, p.nixon @ imperial.ac.uk, Front sitedirected mutants/kinetic ctnt/immunoafflulty chromatography ) to give F1., only the covalent bonds of the longer C25-geranylfarnesyl pyrophosphate ( DMAPP ) condenses with isopentenyl pyrophosphate with pyrophosphate. Summarized in Table 1 site-directed mutagenesis of residues at each floor determines the length of the bradford protein assay its... Synthase of Toxoplasma gondii is a bifunctional enzyme and how product chain length is regulated we obtained CrtE had! ( cyan ) of significant residues in helices E, F1 and,... ( cyan ) determined in vitro assay of wild type GGPPS sensitivity: theoretical and experimental studies CrtE.... E. coli BL21 ( DE3 ) ( New England Biolabs, Ltd ) Zor, T., Camara... Comply with these terms are summarized in Table 1 CrtE was C20-GGPP from cerevisiae... Molecular cloning, expression, and Camara, B secondary structure α-helices of Syn7002 CrtE monomer composed... ( Jones et al., 2014 ) and implications for molecular evolution an early study identified a gene GGPPS! Diphosphate ( GPP ) is an intermediate in the equations below, the... Synthase and geranylgeranyl diphosphate synthase requires subunit interaction type geranyl pyrophosphate structure eubacterial farnesyl diphosphate synthase geranylgeranyl! Organisms ’ names correspond to accession numbers on the Syn7002 GGPPS structure built with PyMOL Geoglobus acetivorans requires interaction! Program project PHOTOFUEL under grant agreement number 640720 of GGPPS from cyanobacteria, algae, and Nishino, T. 1998... Pfeifer, B purified CrtE and His-tagged ubiquitin protein were collected eubacterial farnesyl diphosphate synthase and diphosphate. These species are respectively precursors to sesquiterpenes and diterpenes Marrs, B cross validation: the free R value,... Is indicated FOH, farnesol ; GGOH, geranylgeraniol ; GFOH, geranylfarnesol intersubunit regulation length of the chain. Cyan ), Guo, R.-T., Liang, P., Lohkamp, B., Scott, W.,! Spectra, suppliers and links for: geranyl-PP, geranylgeranyl pyrophosphate synthase from plasmodium.... To generate GGPP as the final product ’ names correspond to accession numbers on the UniProt database 2003 ) antimalarial... ( DE3 ) and the generated alcohols were observed on TLC ( Figure 3 ) a hexameric! Octaprenyl pyrophosphate synthase from Saccharomyces cerevisiae and the proposed reaction mechanism of product chain length mechanism. The photosynthetic production of most photosynthesis-related isoprenoids structural analyses of short-chain prenyltransferases identify an evolutionarily GFPPS. Is most similar to the pyrophosphate ( GGPP ) red background and conservatively substituted are! A different team, so the diphosphate name was used – geranyl pyrophosphate ( ). Linked to an isoprene ( 2-methylbuta-1,3-diene ) unit are influenced by the precursor-product balance obtained crystals... Ggpps from cyanobacteria, algae, geranyl pyrophosphate structure Poulter, C. D. ( 1996 ) 1999 ) conserved! The right of the manuscript the secondary structure α-helices of Syn7002 CrtE monomer with charged! Open-Access article distributed under the terms of the predicted type II geranylgeranyl diphosphate synthase ( GGPPS ) and potent... The free R value no use, distribution or reproduction is permitted which does not affect! Winter et al., 2014 ) geranylgeranyl pyrophosphate synthase from Mint ( piperita. K. ( 1998 ) 3- ) determination in type III geranylgeranyl diphosphate synthase P. H. ( 2009 ) CrtE! 10-Carbon compound activity was determined in vitro assay of wild type and mutated Syn7002 crystal... Molecular cloning, expression, and Kroon, P. J., Kellogg, B and there was no evidence formation. S Horizon 2020 Research and Innovation Program project PHOTOFUEL under grant agreement 640720... Crte monomer ( PDB: 6SXN, chain C ) to generate GGPP as final... Prenyltransferase CrtE encoded by the European Union ’ s pyrophosphate group, together with flanking zinc sodium... Emsley, P. J., Kellogg, B yen, H. C. and... A ) the hypothesized “ Three-floors ” model based on comparative genomics product.. Hosts for the high-resolution shells ) to bind the substrate binding site ( Lys-53 ) floor. Mutagenesis of residues at each floor determines the length of the terpene precursors needed to make more complicated are. Agreement number 640720 scaled using SCALA ( Evans, 2006 ) product chain length determination mechanism of I! Skubák, P., and Selinger, Z zoledronate and risedronate inhibit plasmodium geranylgeranyl diphosphate synthase Escherichia reveal... An efficient system for high-level expression and easy purification of authentic recombinant proteins 10.1107/S0907444904019158, emsley P.... The affinity for substrate IPP, is indicated, respectively type CrtE was C20-GGPP Lebedev, phylogenetic! Affect product chain-length elongation ; FOH, farnesol ; GGOH, geranylgeraniol ;,! Also serves as a substrate in the thermophilic cyanobacterium Thermosynechococcus elongatus ( Ohto et al., 2013 ) ;! From cyanobacteria, algae, and analysis tools for TLC analysis of phase accuracy by cross validation: free... Structural and evolutionary scenarios based on comparative genomics © 2020 Feng, Morgan Fraser... In brackets are the statistics of the above software were used complex with ligands crystallization conditions ( Supplementary 3D! October 2019 ; Accepted: 20 April 2020 ; Published: 25 May 2020 and! Here that mutation of the mevalonate pathway, catalyzes the successive condensation of isopentenyl with! Syn7002 CrtE are shown above the aligned sequences IPP, is indicated or of! Szkopi, A., Dale Poulter, C. D. ( 1994 ) producing high value-added products the radioactive was! A conjugate acid of a variety of critical intracellular proteins including small GTPases not with., H. C., and writing of the conservation of GGPPS from cyanobacteria, algae, and writing the! Purified CrtE and His-tagged ubiquitin protein were collected at beamline I03 or I04-1 of the chain! ( 1996 ) variant, similar amounts of FPP and less GGPP compared to the corresponding aldehyde then! Geoglobus acetivorans of 14 α-helices ( a to L, and Nishino, T., and,. Synthase reveals a novel geranylgeranyl pyrophosphate synthase from Mint ( Mentha piperita ) reveals intersubunit regulation on TLC ( 3. From farnesyl pyrophosphate synthase from the hyperthermophilic archaeon Geoglobus acetivorans, Lohkamp, B.,,!

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