Naturwissenschaften (2009) 96:731–735 DOI 10.1007/s00114-009-0521-1
SHORT COMMUNICATION
Unique animal prenyltransferase with monoterpene synthase activity Anna B. Gilg & Claus Tittiger & Gary J. Blomquist
Received: 28 April 2008 / Revised: 19 February 2009 / Accepted: 21 February 2009 / Published online: 10 March 2009 # Springer-Verlag 2009
Abstract Monoterpenes are structurally diverse natural compounds that play an essential role in the chemical ecology of a wide array of organisms. A key enzyme in monoterpene biosynthesis is geranyl diphosphate synthase (GPPS). GPPS is an isoprenyl diphosphate synthase that catalyzes a single electrophilic condensation reaction between dimethylallyl diphosphate (C5) and isopentenyl diphosphate (C5) to produce geranyl diphosphate (GDP; C10). GDP is the universal precursor to all monoterpenes. Subsequently, monoterpene synthases are responsible for the transformation of GDP to a variety of acyclic, monocyclic, and bicyclic monoterpene products. In pheromone-producing male Ips pini bark beetles (Coleoptera: Scolytidae), the acyclic monoterpene myrcene is required for the production of the major aggregation pheromone component, ipsdienol. Here, we report monoterpene synthase activity associated with GPPS of I. pini. Enzyme assays were performed on recombinant GPPS to determine the presence of monoterpene synthase activity, and the reaction products were analyzed by coupled gas chromatography–mass spectrometry. The functionally expressed recombinant enzyme produced both GDP and myrcene, making GPPS of I. pini a bifunctional enzyme. This unique insect isoprenyl diphosphate synthase possesses the functional plasticity that is characteristic of terpene biosynthetic enzymes of plants, contributing toward the current understanding of product specificity of the isoprenoid pathway.
A. B. Gilg : C. Tittiger : G. J. Blomquist (*) Department of Biochemistry and Molecular Biology/MS 330, University of Nevada, Reno, Reno, NV89557-0014, USA e-mail:
[email protected]
Keywords Pheromone . Mevalonate pathway . Isoprenyl diphosphate . Monoterpene
Introduction Most of our current knowledge regarding terpene biosynthesis comes from research on plants. Terpenes are naturally occurring compounds that function primarily as plant secondary metabolites, constituting the essential oils, resins, and turpentines that play various ecological roles (Wise and Croteau 1999). The isoprenyl diphosphate synthases (IPPSs), geranyl diphosphate synthase (GPPS; EC 2.5.1.1), farnesyl diphosphate synthase (FPPS; EC 2.5.1.10), and geranylgeranyl diphosphate synthase (GGPPS; EC 2.5.1.30) catalyze an electrophilic chain elongation reaction to produce geranyl diphosphate (GDP; C10), farnesyl diphosphate (FPP; C15), and geranylgeranyl diphosphate (GGDP; C20), respectively (Poulter and Rilling 1981). In turn, terpene synthases (TPSs) catalyze the transformation of these linear isoprenyl diphosphate precursors, which often undergo regio- and stereoselective cyclizations and subsequent modifications, into the structurally diverse mono-, sesqui-, and diterpenes (Bohlmann et al. 1998). Bark beetles (Coleoptera: Scolytidae) can be considered similar to plants and unusual among animals given their inherent ability to synthesize monoterpene-derived secondary metabolites. Several bark beetles species produce monoterpene-derived aggregation pheromone components de novo via the mevalonate pathway in the anterior midgut to coordinate the colonization of coniferous trees (Seybold et al. 1995; Hall et al. 2002; Nardi et al. 2002). Upon feeding on host pine phloem, male Ips pini synthesize juvenile hormone (JH) III, which induces the production of the acyclic monoterpene alcohol, ipsdienol (Fig. 1; Tillman
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OPP
IPP + OPP DMAPP
Materials and methods
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Functional expression of recombinant GPPS
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MYRCENE
Geranyl diphosphate synthase/ myrcene synthase
P450 CYP9T2
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IPSDIENOL
Fig. 1 Final steps in the formation of ipsdienol by male I. pini. Geranyl diphosphate synthase (GPPS) catalyzes both the addition of the allylic moiety of DMAPP to IPP to produce GDP and the subsequent conversion of GDP to myrcene in the pheromone biosynthetic pathway
et al. 1998). The transcript levels of mevalonate pathway genes involved in pheromone biosynthesis are coordinately increased in pheromone-producing beetles (Keeling et al. 2004, 2006). Recent research efforts have focused on determining the terminal enzymatic steps of the pheromone biosynthetic pathway. Studies have suggested that acyclic myrcene is involved as a precursor in ipsdienol production in male I. pini; there is an increase in conversion of GDP to myrcene in phloem-fed or JH III-treated male I. pini (Martin et al. 2003) and a cytochrome P450 (CYP9T2) isolated from I. pini hydroxylates myrcene to ipsdienol in pheromone-producing tissue (Sandstrom et al. 2006). A key enzyme in monoterpene biosynthesis is GPPS. GPPS catalyzes the condensation of dimethylallyl diphosphate (DMAPP; C5) and isopentenyl diphosphate (IPP; C5) to produce GDP, the universal precursor to all monoterpenes. Subsequently, monoterpene synthases catalyze the transformation of GDP into a wide array of acyclic, monocyclic, and bicyclic monoterpene products (Wise and Croteau 1999). Although monoterpenes are ubiquitous in nature, GPPSs and myrcene synthases have been characterized in only a few plant species (Tholl 2006; Schmidt and Gershenzon 2008). To date, the only animal GPPS to be reported is from I. pini (Gilg et al. 2005), and there is no previous example of an animal myrcene synthase. Research to elucidate the terminal enzymatic steps of de novo pheromone biosynthesis via the mevalonate pathway in bark beetles has led to a better understanding of how Ips spp. regulate pheromone production, particularly the hydroxylation of myrcene to ipsdienol (Sandstrom et al. 2006). To identify whether GPPS of I. pini is the enzyme responsible for myrcene production, we performed enzymatic assays on the recombinant protein.
A truncated version of GPPS cDNA from I. pini (AY953508) was expressed in Escherichia coli and functionally analyzed for myrcene synthase activity. We chose the truncated version because expression of the full-length clone resulted in extensive inclusion body formation, and molecular modeling studies suggested that the truncated version contained the catalytic domain (see below). The truncated version also resulted in inclusion body formation, but there was enough soluble enzyme for assays. The coding region corresponding to amino acids P51-E416 was amplified by polymerase chain reaction (PCR) by using Pfu Turbo Taq polymerase (Stratagene), an adaptor oligonucleotide (5′ GCGAATTCCAAGTTCTCTGTCC-3′) as the upstream primer designed to create an EcoRI restriction site built into the start site, and a downstream adaptor primer (5′-GCCT CGAGCTTATTCAGGCCTCCTCTT-3′) containing a XhoI restriction site at the terminal stop codon. The PCR product was subcloned into the pET-32c (Novagen) vector that was previously digested with EcoRI and XhoI and transformed into E. coli BL21-Star(DE3)pLysS cells (Invitrogen). The cell culture was grown to an OD600 of 0.5 and then induced in 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 30°C for 2 h. Induced protein levels were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzyme assays and product analysis The expressed recombinant GPPS in crude cell lysates was assayed for monoterpene synthase activity by a protocol adapted from the method described by Bohlmann et al. (1997). Cells were harvested by centrifugation (2,000×g) and frozen at −20°C. The cell pellet was resuspended in assay buffer [(25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.5, 1 mM MnCl2, 100 mM KCl, 3 mM dithiothreitol, 10% glycerol, protease inhibitor cocktail (Sigma)], and sonicated for further cell disruption. To assay for monoterpene synthase activity, 0.5–1 ml of the homogenate (100– 500 μg total protein) was incubated with 5 μM [1-3H]GDP (20 Ci/mmol), or 10 μM [1-3H]IPP (60 Ci/mmol; American Radiolabeled Chemicals) and 50 μM DMAPP (Sigma). For product identification, separate samples of homogenate were incubated with unlabeled 5 μM GDP, or 10 μM IPP and 50 μM DMAPP, and analyzed by gas chromatography–mass spectrometry (GC–MS; see below for conditions). The reactions were placed at 30°C in a shaking water bath for 2–3 h. A 15-min time point was used for samples incubated with [1-3H]IPP and DMAPP. Empty pET32 vector, boiled enzyme, buffer only, and noninduced control groups were run under the same conditions. Each treatment was run in
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triplicate. Following incubation, the reaction mixture was extracted with pentane (3×1 ml) and the combined extract was passed through a 1.5-ml glass column of anhydrous MgSO4 and silica gel (Florisil). Subsequently, the column was washed with pentane (3×1 ml). The eluted hydrocarbon fraction was collected and measured for radioactivity by using a TRI-CARB 2900 TR liquid scintillation analyzer. GPPS activity was determined by using the prenyltransferase acid lability assay previously described in Gilg et al. (2005). The level of GDP production was measured by hydrolysis of GDP to geraniol (GOH) and the amount of GOH represents GDP formed. Products from incubating unlabeled GDP or DMAPP and IPP with expressed enzyme were extracted with pentane or pentane/ether (50:50). Extracts were concentrated under N2 gas. Aliquots were analyzed by coupled GC–MS at the Nevada Proteomics Center (University of Nevada, Reno). A Thermo Finnigan Polaris Q ion trap mass spectrometer was
used with a molecular weight scanning range of 40–180 amu at an ionization potential of 70 eV. A trace gas chromatograph containing a 60-m×0.25-mm (ID)×0.25-μm film thickness Innowax capillary column (J & W Sci Agilent Tech) was programmed at an initial temperature of 40°C, increased to 240°C at 5°C/min with a final hold time of 10 min. The injector was split at a ratio of 100:1 at a temperature of 225°C with a column flow of 1.5 ml He/min. The detector was set at 200°C.
Results Recombinant GPPS converted unlabeled DMAPP plus IPP and, in a separate experiment GDP, to myrcene. The myrcene products formed by GPPS from the initial substrates DMAPP plus IPP and GDP alone had the same retention
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Fig. 2 GC–MS analysis of products formed when recombinant GPPS was incubated with GDP (a) and control samples (b). The trace (c) is a myrcene standard. The mass spectrum of products of GDP incubated
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with GPPS is shown in d, and this spectrum was almost identical to that of the standard myrcene (data not shown)
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time and an identical mass spectrum as a myrcene standard (Fig. 2a–d). Several minor peaks were observed in the mass spectrum of products of GDP, but were not identified. GC– MS analyses showed no myrcene in any of the control groups, including IPTG-treated empty pET32 vector, buffer only, boiled enzyme, and noninduced GPPS samples (Fig. 2b). The expressed recombinant GPPS also showed monoterpene synthase activity by significant (one-way analysis of variance, F1,5 =37.3, P=0.0077) incorporation of 3H-GDP into hydrocarbon products that were present in the pentane extract (Fig. 3a). There was no detectable activity in the IPTG-treated cells with empty pET32 vector or noninduced control samples. GPPS incubated with DMAPP and 3H-IPP as cosubstrates showed the presence of both geranyl diphosphate (as GOH) and terpene hydrocarbon (F1,5 =247, P=0.0005; Fig. 3b). After an incubation of 15–30 min, the amount of geranyl diphosphate was approximately tenfold higher than the level of terpene, as expected if DMAPP and IPP were converted to GDP and then to myrcene.
TERPENE PRODUCTION (DPM +/- S.E.)
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Fig. 3 a Terpene synthase activity measured by the incorporation of 3 H-GDP into terpene hydrocarbons in the cell extract of empty pET32c vector, noninduced sample, and IPTG-induced E. coli expressing GPPS. Bars marked with different letters denote a significant difference. b Relative levels of geranyl diphosphate synthase and monoterpene synthase activity measured by the amount of 3H-IPP incorporated into their respective products in the cell extract of IPTG-induced E. coli expressing recombinant GDPS. No enzyme negative control is included. Bars marked with different letters denote a significant difference in enzyme activity between sample groups
Discussion Here, we report that GPPS of I. pini is a bifunctional enzyme with two distinct enzymatic activities, GPPS activity (Gilg et al. 2005), and myrcene synthase activity. Firstly, the GPPS activity was previously described by Gilg et al. (2005) and secondly, monoterpene synthase. Identifying the enzyme that catalyzes both the electrophilic alkylation of C–C double bond of IPP by DMAPP to produce GDP and the subsequent conversion of GDP to myrcene provides a critical step toward understanding monoterpene-derived pheromone biosynthesis in bark beetles. One- and three-dimensional analyses of the full-length protein consistently predict that it folds into two distinct domains, a short (50–70 residues) N-terminal domain and a prenyltransferase (catalytic) domain (Welch et al., unpublished data). The N-terminal domain is predicted to be largely coil conformation with 30–40% disorder and contains several posttranslational processing motifs. Three different folding algorithms predict that the N-terminal domain has an extended tertiary structure and is remote from the highly conserved substrate binding sites. While we cannot say with absolute certainty the N-terminal domain does not directly affect catalysis, modeling suggests that the N-terminal domain may play an indirect role through posttranslational modification and/or interaction with other cellular constituents. Clearly, the prenyltransferase domain is sufficient for catalysis. When the recombinant enzyme was incubated with DMAPP and IPP as cosubstrates, the level of GDP formed was approximately tenfold higher than the level of myrcene formed. These data suggest that GDP is a free intermediate in myrcene production. The observable difference in the levels of GDP vs. myrcene produced may be due to several factors, including the possibility that GDP exits the binding pocket before its conversion to myrcene. There are several structural and sequence motifs shared between IPPSs and TPSs (Chen et al. 1994; Bohlmann et al. 1998). Both classes of enzymes use a similar reaction mechanism that involves highly reactive, and often multiple, carbocation intermediates to generate their products (Christianson 2007; Keeling et al. 2008). Although I. pini GPPS amino acid sequence has a higher similarity to GGPPSs than to monoterpene synthases of plants (Chen et al. 1994), the similarity in functional character between plant GPPSs and monoterpene synthases makes it plausible that a GPPS could have easily evolved myrcene synthaseassociated activity through functional mutations from a closely-related prenyltransferase gene. Recently Green et al. (2007) demonstrated that α-farnesene synthase of Malus domestica (apple) has prenyltransferase activity, which further illustrates that isoprenoid biosynthetic enzymes have a broad range of substrates and product selectivities.
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To our knowledge, GPPS of I. pini is the first animal prenyltransferase to be shown to have terpene synthase activity. Importantly, the unique bifunctional character of the bark beetle GPPS may have important implications toward understanding the biosynthetic promiscuity of substrate selection and multiproduct formation that is often observed among isoprenoid biosynthetic enzymes. Acknowledgments Supported in part by NSF grant IBN-0719279. This article represents a contribution of the Nevada Agricultural Experiment Station (publication #03087101). We thank the managers of the Whittell Forest (UNR) for allowing us to collect beetles and Jörg Bohlmann for his helpful comments. This research was conducted in accordance with the current laws of the USA. This publication was made possible by NIH Grant Number P20 RR-016464 from the INBRE Program of the National Center for Research Resources.
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