Crystal structure of geranylgeranyl pyrophosphate synthase (crtE) from Nonlabens dokdonensis DSW-6

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Highlights

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    Crystal structure of GGPPS from Nonlabens dokdonensis DSW-6 was determined.

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    NdGGPPS structure is composed 15 α-helices and shows a class I terpenoid synthase fold.

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    NdGGPPS might function as a short-chain prenyltransferase.

Abstract

Isoprenoids comprise a diverse group of natural products with a broad range of metabolic functions. Isoprenoids are synthesized from prenyl pyrophosphates by prenyltransferases that catalyze the isoprenoid chain-elongation process to different chain lengths. We hereby present the crystal structure of geranylgeranyl pyrophosphate synthase from the marine flavobacterium Nonlabens dokdonensis DSW-6 (NdGGPPS). NdGGPPS forms a hexamer composed of homodimeric trimer, and the monomeric structure is composed of 15 α-helices (α1–α15). In this structure, we observed the binding of one pyrophosphate molecule and two glycerol molecules that mimicked substrate binding to the enzyme. The substrate binding site of NdGGPPS contains large hydrophobic residues such as Phe, His and Tyr, and structural and amino acids sequence analyses thereof suggest that the protein belongs to the short-chain prenyltransferase family.

Introduction

Isoprenoids are the most diverse group of products synthesized by bacteria, yeasts, fungi, higher plants, and animals. Isoprenoids include essential metabolites for many biological functions such as membrane fluidity stabilization, free radical exclusion, photoprotection, and hormones precursors [1].

Biosynthesis of isoprenoids occurs over two stages. The first involved the synthesis of universal C5 unit isopentenyl pyrophosphate (IPP) intermediates; the second transforms the IPP into various isoprenoids [2]. IPP biosynthesis occurs via one of two pathways, the mevalonate (MVA) pathway in yeast and mammals, and the methylerythritol phosphate (MEP) pathway in bacteria and plants [3]. The MVA pathway consists of six steps that transform acetyl-CoA to IPP, and then to its isomer, dimethylallyl pyrophosphate (DMAPP), by IPP isomerase (IDI) [4]. The MEP pathway involves a cascade of seven enzymes that transform glyceraldehyde-3-phosphate and pyruvic acid to IPP and then DMAPP [5]. Isoprenoid biosynthesis is catalyzed by prenyltransferases that produce prenyl pyrophosphates of various chain lengths, such as such as geranyl pyrophosphate (GPP, C10, precursor to monoterpenes), farnesyl pyrophosphate (FPP, C15, precursor to sesquiterpenes and triterpenes), geranylgeranyl pyrophosphate (GGPP, C20, precursor to diterpenes) and geranylfarnesyl pyrophosphate (GFPP, C25 precursor to sesterterpenes) (Fig. 1A) [6]. This catalytic reaction by prenyltransferases is initiated by the formation of allyl cations after the removal of pyrophosphate ions to form allyl prenyl pyrophosphate, followed by the addition of IPP with a proton removed from the 2 position. The enzymes then catalyze the transfer of allyl prenyl groups to acceptor molecules with IPP.

Prenyltransferase is classified as both a trans- (C10 to C50) and cis- (C15 to C120) prenyltransferase protein as per structural and stereochemical classification [7,8]. The trans- and cis-prenyltransferases are divided further into short- (C10–20 and C15), medium- (C30–35 and C50-55) and long- (C40–50 and C75-120) chain prenyl pyrophosphate synthases. Trans-prenyltransferases in bacteria include short- and long-chain prenylpyrophosphate synthase homodimers, which produce C10 through C20 by GPP synthase (GPPS) [9], FPP synthase (FPPS) [[10], [11], [12], [13], [14], [15]] and GGPP synthase (GGPPS) [12,16,17], and C40 through C50 by octaprenyl pyrophosphate synthase (OPPS) [18], solanyl pyrophosphate synthase (SPPS) [19,20], and decaprenyl pyrophosphate synthase (DecPPS) [21]. The medium-chain prenylpyrophosphate synthases are heterodimer containing GFPP synthase [22], hexaprenyl pyrophosphate synthase (HexPPS) [23], and heptaprenyl pyrophosphate synthase (HepPPS) [24], which produce C30 through C35. These prenyltransferases ensure that each isoprenoid has a specific number of isoprene units by strict recognition of the prenyl chain length of the allyl substrate and products.

The mechanism whereby the trans-prenyltransferase recognizes the substrate and regulates the reaction and chain length of the product occurs at the hydrophobic cleft near the center of each subunit [14]. The cleft is flanked by two conserved aspartate-rich motifs (DDxxD), called FARM (First Aspartate Rich Motif) and SARM (Second Aspartate Rich Motif). FARM and SARM both recognize the pyrophosphate of the allyl substrate through its essential cofactors, Mg2+ ions; FARM binds the allyl substrate GPP, and SARM binds the homoallyl substrate IPP [25,26]. The final product is made via the addition of IPP to extend the hydrophobic carbon chain from the bottom of the hydrophobic cleft to FARM and SARM. It has been reported that replacement of the aromatic residue inside the cleft by a smaller residue such as alanine or serine results in the synthesis of a longer prenyl chain due to increases in cleft [18,27]. IPP pyrophosphate sites recognize clusters of positively charged residues close to the SARM, and the carbon chain moiety binds to the hydrophobic residues and hydrophobic chain moiety of the allyl substrate [14,28].

Rhodopsin-containing marine flavobacterium Nonlabens dokdonensis DSW-6 is a member the α-proteobacteria class of cytophagia [29,30]. It has recently been isolated from surface seawater and shown to be a unique bacterium of a new genus. In this study, we evaluated the crystal structure of GGPPS from N. dokdonensis DSW-6 (NdGGPPS) to delineate its structure and substrate specificity. By comparing its amino acid sequences and structures to those of other prenyltransferases, we revealed key structural features of NdGGPPS that indicate how the protein recognizes the prenyl chain lengths of allyl substrates and products.

Section snippets

Cloning, expression, and purification

GGPPS gene from N. dokdonensis DSW-6 was amplified with primers: sense, 5- AAACCATGGATGAATACATTGAAATCC-3, and antisense, 5-ATGGGACGTAAAAGCTAACTCGAGAAA-3. The amplified GGPPS gene was inserted into pET30a vector (Merck Millipore). NdGGPPS protein was expressed in E.coli BL21(DE3)T1R strain. The cells were grown in an LB medium containing kanamycin at 37 °C, until absorbance 0.7 at 600 nm. The cells were induced by 1.0 mM IPTG for 20 h at 18 °C. After harvest, cell pellet was resuspended in

Overall structure of NdGGPPS

To investigate the substrate specificity of GGPPS from N. dokdonensis DSW-6 (NdGGPPS), we determined its crystal structure at 2.0 Å resolution (Table 1 and Fig. 1B). The NdGGPPS monomer is predominantly composed of α-helices (α1–α15) (Fig. 2A and B). NdGGPPS is a typical class I terpenoid synthase with an α-helical bundle at its center with surrounded by α-helices [37]. The α1 and α2 helices are perpendicular to the core helices, and the α12 and α13 helices form a lid over the active site. The

Acknowledgements

This work was supported by a Grant from the Next Generation BioGreen 21 Program (SSAC, Code No. PJ01326503 and PJ01326501) from Rural Development Administration, Republic of Korea.

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    These authors contributed equally to this work.

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