Molecular cloning and characterization of a new linalool synthase

ABB Archives of Biochemistry and Biophysics 405 (2002) 112–121 www.academicpress.com

Molecular cloning and characterization of a new linalool synthaseq Anastasia L. Crowell,1 David C. Williams, Edward M. Davis, Mark R. Wildung, and Rodney Croteau* Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA Received 9 May 2002, and in revised form 24 June 2002

Abstract Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol ())-3R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the ())-3R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62–72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a Km value for the natural substrate, geranyl diphosphate, of 56 lM with kcat of 0.83 s
1 . These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: 3R-Linalool synthase; Monoterpene synthase; Mentha citrata; Bergamot mint; Geranyl diphosphate; Essential oil

The monoterpenoids are the simplest class of terpenoids in that they contain only 10 carbon atoms (two isoprene units), and they constitute the major components of the plant-derived essential oils [1]. Most members of this family of natural products are constructed by the monoterpene synthases (cyclases) that catalyze the conversion of geranyl diphosphate, the universal, acyclic C10 intermediate of isoprenoid biosynthesis, to the cyclic parents of the various monoterpene skeletal types [2]. The crucial role of the cyclases in the origin of q The nucleotide sequence reported in this article has been deposited with the GenBank/EMBL database under Accession No. AY083653. * Corresponding author. Fax: 1-509-335-7643. E-mail address: [email protected] (R. Croteau). 1 Present address: Immunex, 51 University Street, Seattle, WA 98101-2936.

the different monoterpene structural groups has stimulated considerable interest in the details of these related enzymatic transformations as a model for the general terpenoid synthase reaction type. Studies with a number of cyclases responsible for the formation of monoterpenes of the p-menthane, pinane, bornane, fenchane, camphane, and thujane families have led to the formulation of a comprehensive proposal for the stereochemical mechanism of cyclization [2,3]. All monoterpene cyclases investigated to date are capable of overcoming the topological impediment to direct cyclization of geranyl diphosphate, imposed by the trans-geometry of the C2–C3 double bond, by way of a preliminary isomerization step that occurs without the formation of detectable free intermediates (Fig. 1). Thus, following productive binding [4], the coupled isomerization–cyclization sequence is initiated by divalent

0003-9861/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 3 - 9 8 6 1 ( 0 2 ) 0 0 3 4 8 - X

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Fig. 1. Mechanistic scheme for the enzymatic conversion of geranyl diphosphate to linalool or to cyclic monoterpenes. Formation of cyclic products from the a-terpinyl cation (b) requires preliminary isomerization of geranyl diphosphate to either (3R)- or (3S)-linalyl diphosphate; the formation of (3R)- or (3S)-linalool involves simple trapping by water of the initially formed, acyclic carbocation (a). OPP denotes the diphosphate moiety, and M2þ is the divalent metal ion required for the two ionization steps of the normally coupled isomerization–cyclization sequence. The structures of limonene, a-terpineol, and myrcene are also illustrated.

metal ion-dependent ionization of geranyl diphosphate, with ensuing syn migration of the diphosphate moiety of the ion pair, to provide the bound tertiary allylic intermediate linalyl diphosphate. In this ionization–isomerization step, which removes the topological barrier to cyclization, the first formal chiral center is introduced at C3 [i.e., either (3R)- or (3S)-linalyl diphosphate is generated from the corresponding left-handed and righthanded helical folding of the geranyl substrate]. After rotation about the C2–C3 bond to afford the cisoid, anti– endo-conformer, linalyl diphosphate is itself ionized with C6–C1 ring closure to provide the corresponding (4R)or (4S)-a-terpinyl carbocation  pyrophosphate anion pair, from which all cyclic monoterpenes derive. These early reaction steps appear to be common to all monoterpene cyclizations, with subsequent steps involving termination of the reaction either by deprotonation or by nucleophile capture or further electrophilic cyclization to the remaining double bond, hydride shift, or Wagner– Meerwein rearrangement before termination. Although strong suggestive evidence for the intermediacy of linalyl diphosphate in cyclase catalysis has accumulated [5–13], all previous efforts to observe directly this product of the mandatory isomerization step, for example by utilizing noncyclizable substrate analogs

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[5,9,13], have failed, presumably because the highly reactive tertiary allylic system is generated at the same active site where the subsequent ionization and cyclization occur (i.e., in this tightly coupled sequence, the binding and reaction of the intermediate generated at the active site are greatly favored over dissociation). Preliminary attempts to dissect the cryptic isomerization step of the normally coupled reaction sequence by directed mutagenesis of extant monoterpene cyclases [14] have also met with limited success. An alternate approach to examining the isomerization step of the reaction is the evaluation of monoterpene synthases that carry out only this partial reaction. No linalyl diphosphate synthase has yet been described; however, two linalool synthases, capable of transforming geranyl diphosphate to the tertiary allylic alcohol as the sole product have recently been cloned. The 3S-linalool synthase of Clarkia breweri is involved in floral scent production in this species [15]. A detailed analysis of this linalool synthase gene suggests that it is a composite structure of recent origin [16], which more closely resembles a diterpene synthase than a monoterpene synthase [17]. The 3R-linalool synthase cDNA recently isolated from Artemisia annua does resemble in primary sequence other monoterpene synthases (41–42% identical to monoterpene synthases of the mint family at the deduced amino acid level) [18]. However, the failure to detect linalool in Artemisia tissues in which the gene was expressed, coupled to the kinetic performance of the enzyme, called into question the physiological relevance of linalool production in planta and suggested that geranyl diphosphate may not be the natural substrate, nor linalool the natural product, of this ‘‘linalool synthase’’ in vivo [18]. For comparative studies of linalool synthase and monoterpene cyclases to be informative, a bona fide linalool synthase more closely related to extant cyclases was required. Here we report the isolation of a cDNA from Mentha citrata Ehrh. which encodes a kinetically competent linalool synthase that resembles monoterpene cyclases of the mint family sufficiently closely to permit structural and mechanistic inference.

Materials and methods Plant materials, reagents, and general protocols Bergamot mint (designated as Mentha  piperita L. nothosubsp. citrata) and lavender mint (designated as Mentha  piperita L. nothosubsp. lavandula) plants were purchased from Nichols Garden Nursery (Albany, OR) and were grown and vegetatively propagated in the greenhouse under conditions previously described for other mint species [19]. English lavender (Lavandula angustifolia) seed was purchased from Richters (Goodwood, Ont., Canada) and similarly raised in the greenhouse.

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The preparation of geranyl diphosphate [20], [1- H]geranyl diphosphate (100 Ci/mol) [21], ())-3Rand (+)-3S-linalyl diphosphate (both >95% ee), and ())3R-[1-3 H]linalyl diphosphate (34 Ci/mol, 95% ee) [6] have been previously described. ())-3R-Linalool (95% ee) and (+)-3S-linalool (99% ee) were from our own collection, as were other terpenoid standards. All other biochemicals and reagents were purchased from Sigma Chemical or Aldrich Chemical, unless otherwise noted. DNA fragments were gel purified using Micropure Separators (0.22-lm Millipore filter; Amicon) with nebulizer attachments or with the QIAquick Gel Purification Kit (Qiagen, Valencia, CA). Routine cloning of PCR products as accomplished with the TOPO-TA Cloning Kit (Invitrogen, San Diego, CA), using the pCR 2.1TOPO vector and Escherichia coli One Shot TOP10F0 competent cells. Plasmids were prepared from liquid cultures using the Miniprep Express Matrix (BIO 101, La Jolla, CA) and procedures recommended by the manufacturer. Primers were synthesized by Life Technologies (Gaithersburg, MD), and automated DNA sequencing was conducted at the Washington State University Laboratory for Biotechnology and Bioanalysis. 3

Essential oil analysis Freshly picked shoot tips (in which essential oil biosynthesis is most rapid [22]), leaves (all ages), or flowers were placed in a Teflon-sealed glass test tube containing 2 ml of pentane and shaken at room temperature for 45 min. The extract was concentrated under a stream of N2 and subjected directly to GC-MS analysis under conditions described previously [23]. Procedures for resolution of monoterpene enantiomers by chiral phase capillary GC have also been described elsewhere [13]. RNA extraction and cDNA synthesis RNA was isolated from immature Bergamot mint leaves using a literature procedure [24]. In brief, shoot tips were pulverized in liquid N2 with a mortar and pestle, and the resulting powder was steeped at 65 °C in extraction buffer containing the RNase inhibitor aurin tricarboxylic acid [25]. After filtration through Miracloth (Calbiochem, San Diego, CA) and initial precipitation with 1.4 M potassium acetate, the RNA was precipitated (overnight, 4 °C) by addition of LiCl to 2 M and was further purified by washing with 3 M sodium acetate and by phenol/ chloroform/isoamyl alcohol extraction. cDNA synthesis reactions contained 1 lg total RNA, 2 ll of 10 first-strand synthesis buffer (Stratagene, La Jolla, CA), 0.5 mM each dNTP, 200 U of M-MLV reverse transcriptase (Promega, Madison, WI), and 37.5 pmol oligo(dT) anchor primer (50 -GAC CAC GCG TAT CGA TGT CGA CTT TTT TTT TTT TTT TT-30 ; from Boehringer Mannheim) (for 30 -RACE, see below) or

12.5 pmol 945R1 primer (50 -CTT TTA GAA TGT GGT ACG CCA ACT CGG-30 ) (for 50 -RACE, see below). The 20-ll reaction mixture was incubated at 42 °C for 1 h, followed by 10 min at 65 °C to inactivate reverse transcriptase. 30 -RACE A series of primers designed for consensus sequences of extant monoterpene synthases from the mint family [17,26,27] were used in 30 -RACE reactions with bergamot mint cDNA template. The 50-ll reactions contained template (diluted 1:20), 3 mM MgCl2 , 200 lM each dNTP, 1 U Taq DNA polymerase with recommended buffer (Life Technologies), 10 pmol of gene-directed primer, and 12.5 pmol of anchor primer (50 -GAC CAC GCG TAT CGA TGT CGA C-30 ) (Boehringer Mannheim). Reactions were carried out at three annealing temperatures (45, 55, and 65 °C) for 30 cycles at 94 °C for 45 s, annealing temperature for 45 s, and 72 °C for 2 min. An amplicon of the predicted size was observed at all annealing temperatures with primer 945NF4 (50 -CCG AGT TGG CGT ACC AGA TTC TAA AAG-30 ), and this product was cloned and sequenced; about 15% of the transformants harbored a sequence similar to those of other monoterpene synthases in the database [28], particularly those of the Lamiaceae [29,30]. 50 -RACE The 50 terminus of the clone identified by 30 -RACE with primer 945NF4 was acquired by preparing cDNA primed with the corresponding reverse primer (designated 945R1 as described above). To the purified product, 200 lM dATP was added and the mixture was heated to 95 °C for 3 min and chilled on ice. Terminal deoxynucleotidyl transferase (15 U; Life Technologies) and the recommended amount of 10 buffer were then added, and the mixture was incubated at 37 °C for 20 min and then at 75 °C for 10 min to inactivate the transferase. This purified product (1 ll) was used directly as template for PCR amplification using 10 pmol each of a nested reverse primer (945R2) and forward primer FPR945 (50 -ATG TGT ACT ATT ATT AGC G-30 ), employing Taq DNA polymerase, 3 mM MgCl2 , and 200 lM each dNTP with 35 cycles at 94 °C for 45 s, 45 °C for 45 s, and 72 °C for 120 s. The single 1.1-kb amplicon obtained was cloned, and sequence analysis showed that most of the transformants contained the 50 terminus of the clone acquired by 30 -RACE; no other monoterpene synthase-like sequences were observed. PCR amplification of a pseudomature sequence For functional characterization of the encoded monoterpene synthase, a pseudomature version of the

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protein was prepared from the defined clone by deleting the 50 codons specifying the plastidal transit peptide characteristic of this enzyme type [17,26]. Thus, PCR primers were designed to yield a cDNA that translated an N-terminally truncated form in which the targeting sequence was eliminated and a new starting methionine was installed immediately upstream of a threonine followed by the conserved, tandem arginine pair (R69R70) that signifies the approximate processing site of other monoterpene synthases [26,31]. To exploit the expression vector pSBET [32], which encodes a tRNA for rare arginine codon usage in E. coli that is common in higher plants, a multistep (sticky-end) PCR protocol [33] was used because of the limited cloning sites in this vector and the presence of an internal restriction site in the insert cDNA. With the 30 -RACE cDNA as template, forward primers (50 -TAT GAC CCG ACG TTC CGG AAA CTA CC-30 and 50 -TGA CCC GAC GTT CCG GAA ACT ACC ACC-30 ) were used to generate an NdeI overhang at the new starting methionine (i.e., E67M, upstream of R69R70) and reverse primers (50 -CTC AGA CAT ATG GCT TGA ACA GCA AGT TCG-30 and 50 -GAT CCT CAG ACA TAT GGC TTG AAC AGC AAG-30 ) were employed to create a BamHI overhang just downstream of the stop codon. The annealed sticky-end PCR product resulting from this protocol [33] was directionally ligated into NdeI/BamHI-digested pSBET and transformed into E. coli XL2-Blue cells. Plasmid was purified from the resulting transformants, and the insert was verified by sequencing before transformation into E. coli BL21(DE3) for expression studies. To compare the truncated version of the enzyme to the preprotein form, the full-length cDNA was prepared for subcloning into pSBET by PCR amplification using Pfu DNA polymerase with forward primer 50 -CAT ATG TGT ACT ATT ATT AGC GTA AAT C-30 (which adds an NdeI site at the starting methionine) and reverse primer 50 -GGA TCC TCA GAC TAT GGG CTT GAA CAG C30 (which adds a BamHI site immediately downstream of the stop codon and eliminates the internal NdeI site near the 30 terminus). The resulting amplicon was first cloned into pCR4Blunt-TOPO (Invitrogen), then digested with NdeI and BamHI, and finally ligated into similarly digested pSBET for expression. Bacterial expression, enzyme purification, and assay A 50-ml culture of Luria–Bertani broth supplemented with 50 lg kanamycin/ml was inoculated with a single colony of transformed E. coli BL21(DE3) cells and incubated at 37 °C with shaking at 250 rpm to a cell density of 0:7A600 . The temperature was reduced to 20 °C and the culture allowed to equilibrate before induction by addition of isopropyl b-D -thiogalactoside to 1 mM. After overnight incubation at 20 °C, cells were harvested by centrifugation at 3000g for 15 min and the pellet was re-

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suspended in 1 ml assay buffer containing 25 mM Tris–Cl buffer (pH 7.5), 5% (v/v) glycerol, 1 mM dithiothreitol, 10 mM MgCl2 , and 1 mM MnCl2 . Cells were frozen in liquid nitrogen, rapidly thawed in warm water for three cycles, and then sonicated for 1 min with a small probe at medium power (VirTis, Gardiner, NY). After centrifugation of the lysate at 40,000g for 10 min, the resulting supernatant (employed as the crude enzyme source) was used in preliminary studies to define the enzyme product. A 100-ll aliquot of the clarified supernatant was diluted into 3 ml of assay buffer (described above) in a Teflonlined screw-capped tube to which 50 lM of geranyl diphosphate, ())-3R-linalyl diphosphate, or (+)-3S-linalyl diphosphate was added. The mixture was overlaid with 1 ml of hexane to trap volatile products; following incubation for 2 h at 30 °C, vigorous mixing and brief centrifugation were done to separate phases, the hexane overlay was removed, and the contents were analyzed. For more detailed characterization, a 1-liter culture was prepared under the same conditions described above, and the harvested cells were resuspended and sonicated in 10 ml of buffer A [25 mM 3-(N-morpholino)-2-hydroxypropanesulfonic acid (Mopso) (pH 6.8), containing 5% (v/v) glycerol and 1 mM dithiothreitol]. After centrifugation at 40,000g for 30 min, the clarified supernatant was applied to a 2:5  10-cm column of Odiethylaminoethyl cellulose (DE52; Whatman) which was equilibrated with buffer A. After washing with 50 ml of buffer A, the matrix was eluted with a 500-ml linear gradient from 0 to 500 mM NaCl in buffer A. A protein fraction which eluted at about 300 mM NaCl contained the bulk of the linalool synthase activity, and this material was applied to a 1  10-cm column of ceramic hydroxyapatite (Bio-Rad) which was equilibrated with buffer A. After washing with 50 ml of buffer A, the column was eluted with a 500-ml linear gradient from 0 to 100 mM potassium phosphate in buffer A. Fractions containing linalool synthase (eluting at about 60 mM) were combined and loaded directly onto a 2  10-cm column of Hi-Q strong anion-exchange matrix (BioRad) which was equilibrated with buffer A. After washing with 50 ml of buffer A, the column was eluted with a 500-ml linear gradient from 0 to 500 mM NaCl in buffer A. The fractions containing linalool synthase (eluting at about 200 mM NaCl) were combined and desalted by repeated concentration and dilution with buffer A using a Centriprep YM-50 centrifugal concentrator (Millipore); this material was used as the enzyme source for all subsequent studies. The course of the purification was monitored by enzyme assay (liquid scintillation counting of the product generated from [1-3 H]geranyl diphosphate) and by SDS– PAGE [34]. Total protein content was determined by the Bradford method [35] using recombinant limonene synthase as standard [31]. The proportion of linalool synthase present in the preparation was determined by

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densitometry (Gel Doc 2000 system; Bio-Rad) of the SDS–PAGE gels after Coomassie staining as described previously [29]. These preparations afforded the linalool synthase at 82–85% purity and at a concentration of about 0.4 mg protein/ml.

nonlinear regression analysis (R ¼ 0:94–0:96) using the Enzyme Kinetics Package (Trinity Software).

Product analysis

Selection of source tissue

Hexane extracts from the enzyme assays with unlabeled substrates were analyzed directly by cool on-column injection, capillary gas chromatography-mass spectrometry [23] using a 5% phenyl-substituted ZB-5 column (30 m0.25 mm i.d. with 0.25-lm film; Phenomenex), with temperature programming at 20 °C/min from 40 to 300 °C. The product was identified by comparison of retention time and mass spectrum to those of the authentic standard. For the separation of linalool enantiomers, the hexane extract was first passed over a small column of silica gel surmounted by MgSO4 and then eluted with diethyl ether. The eluate was analyzed by GC-MS as above but on a b-cyclodextrin chiral capillary column (RTbDEXsm; Restek, Bellefonte, PA) using split injection at 150 °C on a Hewlett-Packard 5890 chromatograph with temperature programming from 70 to 120 °C at 5 °C/min and then to 200 °C at 20 °C/min. Confirmation of identity was provided by comparison to authentic standards of 3R- and 3S-linalool.

Since much of our prior work on monoterpene biosynthesis has been focused on members of the mint (Lamiaceae) family, from which cDNAs encoding a range of monoterpene cyclases have been isolated from the genera Mentha, Salvia, and Perilla [29,30,36], a gene for linalool synthase was sought from this source. The monoterpene components of the essential oils of several potentially useful species were screened by extraction of immature leaves from greenhouse material followed by GC-MS analysis. Bergamot mint (sold as Mentha  piperita L. nothosubsp. citrata) was shown to contain 62.1% linalool and 37.9% linalyl acetate in the essential oil (Fig. 2A). Lavender mint (sold as Mentha  piperita L. nothosubsp. lavandula) was shown to contain 20% linalool, 67% linalyl acetate, and 13% 1,8cineole in the essential oil, and English lavender (Lavandula angustifolia) was shown by similar means to produce 24% linalool, 44% linalyl acetate, 3% 1,8-cineole, and a range of monoterpene olefins. The lavender mint and bergamot mint plants appear to be Mentha citrata (now M. aquatica) types [37] originally selected by Todd and Murray [38] and designated EO661 and EO665, respectively. The present oil analyses are consistent with prior descriptions of these strains [39]. Because linalyl acetate is derived from linalool, bergamot mint appeared to produce but a single monoterpene synthase product (GC-MS analysis of linalool on a chiral capillary column showed the presence of ())-3Rlinalool at 98% and the (+)-3S-enantiomer at 2%; see below). Bergamot mint was therefore used as a source of mRNA in a homology-based cloning effort to acquire the 3R-linalool synthase.

Enzyme characterization For the determination of divalent metal ion requirement, assays at substrate saturation were carried out in buffer A (without divalent cation) and with this same buffer in the presence of MnCl2 (concentrations from 0.1 to 10 mM) or MgCl2 (concentrations from 1 to 50 mM) or both cations. For the determination of pH optimum, assays at substrate saturation were performed in 4morpholineethanesulfonic acid (Mes), pH 5.5, 6.0, and 6.5, Mopso pH 6.0, 6.5, 7.0, and 7.5, and Tris–Cl, pH 7.5, 8.0, 8.5, and 9.0. For initial kinetic analyses, triplicate assays using [1-3 H]geranyl diphosphate as substrate (concentrations from 1 to 50 lM) were performed as described previously for limonene synthase [31]. However, preliminary results revealed an unusually high kcat and Km for this enzyme which required modification of the standard protocol. Triplicate assays were performed with 2 ng enzyme in a total volume of 15 ll of 25 mM Mopso (pH 6.8), containing 1 mM dithiothreitol, 20 mM MgCl2 , and [1-3 H]geranyl diphosphate at concentrations ranging from 3 to 240 lM. The assay mixtures were overlaid with 100 ll hexane, incubated at 30 °C for 10 min, and then vortexed briefly and 50 ll of the overlay was removed for liquid scintillation counting. Kinetic parameters were calculated by Lineweaver–Burke plotting and

Results and discussion

cDNA isolation and characterization Based on the assumption that the linalool synthase from mint, which catalyzes formation of an acyclic monoterpenol by a mechanistically simple reaction cycle, was a ‘‘defective’’ monoterpene cyclase from this plant family, an homology-based PCR cloning strategy was devised using primers designed from consensus sequences of extant monoterpene cyclases from the Lamiaceae [17,26,27]. This approach, by overlapping 30 RACE and 50 -RACE with confirming full-length amplification, yielded a single cDNA of 1818 nt which encodes a deduced protein of 606 amino acids (aa) with a mass of 70.5 kDa and calculated pI of 5.8. This cDNA (Accession No. AY083653) does resemble monoterpene

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A

B

C

D

Fig. 2. (A) Capillary GC-MS analysis (total ion chromatogram) of the essential oil extract of Bergamot mint leaves demonstrating the presence of linalool (Rt ¼ 5:88 min; 62.1%) and of linalyl acetate (Rt ¼ 7:04 min; 37.9%). (B) Capillary GC-MS analysis (total ion chromatogram) of the reaction product of the recombinant enzyme with geranyl diphosphate as substrate. The retention time and mass spectrum (C) of the sole product of the reaction are identical to those of authentic linalool. (D) Chiral phase, capillary GC analysis (flame ionization detection) of the reaction product of the recombinant enzyme with geranyl diphosphate as substrate. The presence of the 3R-antipode (98%) and of the 3S-antipode (2%) were determined by coincidence of retention time with that of the corresponding, optically pure standard.

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cyclases of the Lamiaceae (Mentha [29], Perilla [36], Salvia [30]) in exhibiting 61–72% sequence identity at the nucleotide level, but this presumptive linalool synthase sequence differs substantially from that of the 3S-linalool synthase from Clarkia [16] of the Onagraceae (41% identity) and that of the 3R-linalool synthase from Artemisia [18] of the Asteraceae (52% identity). The cDNA appeared to encode a typical N-terminal plastidial targeting sequence, which is anticipated for a monoterpene synthase [26,40], and the expected DDXXD element in the C-terminal domain (aa363– aa367), which plays an essential role in substrate binding and ionization [14]. The cDNA also encodes tandem arginine residues near the N terminus (R69R70), which are highly conserved in the monoterpene cyclases [26] and are necessary for the conduct of the initial isomerization of geranyl diphosphate (to bound linalyl diphosphate) that must precede the normally coupled cyclization catalyzed by this enzyme type [31]. Comparison of the presumptive linalool synthase sequence with those of the closely related limonene synthase from Mentha [29] (which catalyzes isomerization and monocyclization) and sabinene synthase from Salvia [30] (which catalyzes isomerization and bicyclization with intervening hydride shift) revealed a substantial number of conserved residues; however, the putative linalool synthase contained a three-residue deletion (between aa589 and aa590) relative to the two monoterpene olefin synthases. Thus, circumstantial evidence suggested that the homology-based approach to acquisition of the linalool synthase cDNA had been successful. Nevertheless, confirmation of the clone by functional expression was required. cDNA expression For the purpose of functional expression, a 50 -truncated version of the cDNA was prepared in which codons specifying the N-terminal transit peptide were deleted and an ATG was appended to install a new starting methionine immediately upstream of the highly conserved arginine pair to initiate translation as ME67T68R69R70. Such ‘‘pseudomature’’ forms generally afford recombinant monoterpene synthases that are more soluble and readily purified and that closely resemble the native enzymes in kinetic properties [31]. pSBET [32] was chosen as the expression vector for use in E. coli BL21(DE3) cells because this plasmid additionally encodes a tRNA for rare arginine codon usage in the bacterial host that is common in higher plants; such precaution often prevents premature termination of translation [29,31]. Expression under standard conditions, followed by protein isolation, assay, and SDS– PAGE, demonstrated the presence of an activity in the soluble enzyme fraction that was capable of converting [1-3 H]geranyl diphosphate to radiolabeled, hexane-sol-

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uble products (this activity was negligible in enzyme preparations from ‘‘empty’’ pSBET vector controls) and the presence of the predicted 63-kDa protein band on the corresponding SDS–PAGE gel (that was also absent in enzyme preparations from empty vector controls). Immunoblotting using polyclonal antibodies directed against the limonene synthase from Mentha spicata [41] failed to detect the presumptive linalool synthase and, thus, provided no supporting evidence for the identity of the target clone as a monoterpene synthase. However, the recombinant enzyme was inactive with the sesquiterpene precursor farnesyl diphosphate and the diterpene precursor geranylgeranyl diphosphate, thus eliminating the possibility that the encoded protein was a sesquiterpene or diterpene synthase. Product analysis Initial studies using the crude recombinant enzyme preparation were directed to the identification of the product(s) derived from the universal monoterpene precursor geranyl diphosphate. Capillary GC-MS analysis of the hexane-soluble products generated from geranyl diphosphate at apparent saturation (150 lM) demonstrated linalool to be the sole product of the enzymatic reaction (Figs. 2B and C; note the absence of geraniol in 2B indicating that the preparation was free of phosphatase activity). Boiled controls yielded only trace levels of linalool derived by (nonenzymatic) solvolysis of the substrate during the course of the assay [42,43]. Chiral phase capillary GC analysis (b-cyclodextrin column) revealed (Fig. 2D) the enzyme product to be ())-3R-linalool (96% ee), nearly the same enantiomer distribution determined for linalool of the extracted leaf oil. The lower enantiomeric purity reported in the literature for steam-distilled oils from this species ( 88% ee) is typical for essential oils containing high levels of linalyl acetate and results from partial racemization by thermally induced solvolysis of the ester during distillation [44]. These results confirmed that a cDNA encoding ())-3R-linalool synthase had, in fact, been isolated from M. citrata. Although the full-length version of the enzyme (70.5 kDa including the transit peptide) also produced ())-3R-linalool as the only product, this form was less efficiently expressed as a soluble protein and was less active than was the truncated version which was employed in all subsequent studies. Identical assays with ())-3R-[1-3 H]linalyl diphosphate as substrate at apparent saturation (150 lM) gave about half the rate of conversion to hexane-soluble products as that observed with geranyl diphosphate. Most monoterpene cyclases utilize the tertiary allylic isomer (which is not constrained with respect to cyclization) at two to three times the rate observed with geranyl diphosphate [2,3]. GC-MS analysis of the products generated from ())-3R-linalyl diphosphate

showed a moderate reduction in the abundance of linalool (71% of the total mix) accompanied by the formation of several products not observed with geranyl diphosphate as substrate, including the cyclic alcohol aterpineol (20%), the linear, achiral olefin myrcene (5%), and the monocyclic olefin limonene (2.5%). Chiral phase capillary GC analysis demonstrated these products to be ())-3R-linalool (76% ee), (+)-4R-a-terpineol (80% ee), and (+)-4R-limonene (56% ee) (see Fig. 1 for structures). Identical assays with (+)-3S-linalyl diphosphate at apparent saturation showed this antipode to be a poor substrate ( 10% of the rate of conversion of geranyl diphosphate), and only two products were observed, linalool (22%) and a-terpineol (78%). Chiral phase separation, as before, demonstrated these products to be ())-4S-a-terpineol (80% ee) and nearly racemic linalool (8% ee for the ())-3R-antipode). The observation that both enantiomers of linalyl diphosphate yielded cyclic products (in addition to linalool) that were not formed from geranyl diphosphate, coupled to the absence of such cyclic products in the leaf essential oil, indicate that linalyl diphosphate is not likely to be an intermediate in the formation of linalool from geranyl diphosphate and that linalyl diphosphate is not likely to be formed at all in vivo by this enzyme. Enzyme purification and characterization The crude recombinant enzyme preparation was separated by a combination of hydroxyapatite and anion-exchange chromatography to yield highly purified linalool synthase (>82% purity by SDS–PAGE, and free of competing activities) that was used for characterization studies. The pH optimum for activity was determined in Mes, Mopso, and Tris–Cl ranging in pH from 5.5 to 9.0 and using geranyl diphosphate as substrate. The optimum was shown to be near pH 6.5 (with halfmaximum velocity at pH 7.5; the half-maximum velocity on the acid side was near pH 5.5 but could not be determined accurately because of solvolytic decomposition of the substrate to linalool with decreasing pH). This optimum pH value is fairly typical for monoterpene synthases from angiosperms [3]. In the absence of divalent metal ion, linalool synthase was essentially inactive (with any test substrate), but activity could be restored by the addition of either Mg2þ or Mn2þ . The maximum activity achieved by addition of either metal ion was nearly identical, with a saturating concentration of Mg2þ at about 20 mM (similar to that for other monoterpene synthases) and a saturating concentration of Mn2þ at about 0.2 mM (lower than the 1–2 mM saturation generally observed with related synthases) [3]. Steady state kinetic analysis of the purified recombinant enzyme yielded a Km value for geranyl diphosphate of about 25  6 lM with kcat of 0:24  0:02 s
1 ; both are

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relatively high values for a monoterpene synthase [3]. It was observed that, during the purification of recombinant linalool synthase, both Km and kcat values decreased with the removal of contaminating host protein. While this alteration in catalytic properties could result from degradation of the enzyme during purification, the simultaneous change in both Km and kcat suggested that interaction (or lack thereof) between the synthase and the host protein(s) might be responsible for the observed effect. To test this possibility, the combined E. coli protein that had been removed upon purification (this material was itself catalytically inactive) was added back to the purified synthase and the kinetics were reevaluated; both Km and kcat were increased over twofold. To examine the specificity of this phenomenon, the influence of bovine serum albumin, cytochrome c, and carbonic anhydrase on the purified linalool synthase was determined, and all three of these unrelated proteins were found to promote a similar increase in kinetic constants, yielding maximum values of Km ¼ 56  5 lM and kcat ¼ 0:83  0:08 s
1 at equimolar concentrations of foreign protein and linalool synthase (i.e., 40 nM protein at substrate concentrations ranging from 2 to 150 lM geranyl diphosphate). Other additives tested (NaCl, KCl, Tween 20, and glycerol at a range of concentrations) had no discernable influence on linalool synthase activity. Because the activity of the recombinant enzyme was observed to degrade more rapidly during storage when the protein was dilute, experiments were conducted to determine whether the added foreign protein prevented loss of linalool synthase activity during storage and subsequent assay. These results (data not shown) did demonstrate a stabilizing influence of the foreign protein, but this effect was largely independent of the kinetic influence resulting from these protein– protein interactions. Although this phenomenon is probably not relevant in situ, it is an important consideration for the operational use of the purified enzyme in biotechnological applications.

Conclusions A cDNA encoding ())-3R-linalool synthase from Mentha was successfully isolated by a homology-based cloning strategy founded upon the assumption that the sequence for this acyclic monoterpenol synthase would resemble those of monoterpene cyclases from the mint family that catalyze more complex reaction types. At the nucleotide level, the sequence of 3R-linalool synthase was shown to be more similar to phylogenetically related monoterpene cyclases (62–72% identity) than to extant, but phylogenetically distant, linalool synthases (41–52% identity). In most properties, the recombinant linalool synthase is also similar to the monoterpene cyclases; however, both Km and kcat values are notably higher

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than those of most other enzymes of this type [3], likely reflecting differences in the active site of linalool synthase that underlie the production of the acyclic product by this apparently ‘‘defective’’ monoterpene cyclase. The mechanism of linalool synthase is presumed to involve binding of the geranyl substrate, without the requirement to orient the substrate olefinic chain in anti– endo-conformation required for cyclization [3], followed by metal ion-assisted ionization of the diphosphate ester, with stereoselective capture by water of the resulting allylic carbocation (Fig. 1). Kinetic evaluation of 3Rand 3S-linalyl diphosphate as inefficient alternate substrates appears to rule out the tertiary allylic diphosphate as an intermediate in the reaction, but these studies unexpectedly revealed that, when the geometric constraint to cyclization was removed (i.e., the trans-2,3double bond of geranyl diphosphate), the linalool synthase was capable of catalyzing cyclization to a-terpineol (and, much less efficiently, to limonene). It is notable that, in these reactions, 3R-linalyl diphosphate gave rise to 4R-a-terpineol (20% of product mix) and 4R-limonene (2.5%) and that 3S-linalyl diphosphate gave rise to 4S-a-terpineol (78% of product mix). These stereochemical correlations are consistent with cyclization via the anti–endo-conformation of the substrate, as is typical of monoterpene cyclases [2,3]. However, the linalool derived from 3R-linalyl diphosphate was largely the 3R-enantiomer (76% ee) while that derived from 3Slinalyl diphosphate was nearly racemic, indicating that water capture readily occurs on the same face of the C1– C2–C3 delocalized p-system from which the diphosphate departs and not from the opposite side to which C6–C1 bond formation takes place in the corresponding cyclization. Thus, it can be inferred that backside attack by water at C3 of linalyl diphosphate (i.e., with inversion of configuration) is disfavored, likely by shielding with the substrate alkyl chain bound in the active site, and that, upon ionization, the diphosphate moiety must be sufficiently displaced (or lost) to expose this same face to solvent capture. It is additionally noteworthy that, even with linalyl diphosphate as substrate, from which cyclization is possible, over 90% of the products generated (linalool plus a-terpineol) arise by water capture of the corresponding carbocations. The results of comparing geranyl diphosphate and linalyl diphosphate as substrates for linalool synthase indicate that this enzyme is incapable of catalyzing the isomerization step as a prelude to the coupled cyclization conducted by the monoterpene cyclases and that the active site of linalool synthase is more accessible to water than that of most monoterpene cyclases in which water is excluded to prevent premature solvent capture of intermediate carbocations. The stereospecific reaction catalyzed by 3R-linalool synthase of M. citrata can thus be formulated (Fig. 3) as involving binding of geranyl diphosphate as the left-handed conformer (unlike 4S-limonene

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A

Fig. 3. Stereochemical scheme for the conversion of geranyl diphosphate to ())-3R-linalool by M. citrata ())-3R-linalool synthase. OPP denotes the diphosphate moiety, and M2þ is the divalent metal ion.

synthase of M. spicata and M. piperita [45,46]) to prevent backside water attack and promote the approach of water from the ‘‘top side’’ to form the 3R-enantiomer upon ionization and displacement of the diphosphate. Sequence comparisons of linalool synthase with limonene synthase and sabinene synthase, two highly conserved monoterpene synthases which produce cyclic olefins, identified a three-amino acid deletion in linalool synthase relative to the olefin cyclases (occurring between aa589 and aa590 in linalool synthase). The analogous region in tobacco epi-aristolochene synthase (TEAS), a sesquiterpene olefin cyclase for which the crystal structure has been solved [47], comprises the J/K loop which upon substrate binding serves as an active-site lid to prevent water access to the subsequently formed carbocationic intermediates. Comparison of models of the active sites of linalool synthase, sabinene synthase, and limonene synthase (using the TEAS structure as scaffold) clearly shows a major change in the position of this loop in linalool synthase (Fig. 4A) relative to the two olefin cyclases (Figs. 4B and C). The positioning of this loop is consistent with this structural feature performing a role in the monoterpene olefin synthases similar to that in TEAS, whereas in linalool synthase, the three-residue deletion results in substantial outward displacement of the J/K loop to provide a ready means of water access to the active site upon substrate ionization. A direct consequence of the J/K loop displacement in linalool synthase is the positional changes of a conserved aspartate and an asparagine (histidine in the olefin cyclases) relative to the diphosphate (Fig. 4). Thus, D587 in linalool synthase is considerably removed from the diphosphate moiety (relative to D577 and D568 in limonene synthase and sabinene synthase, respectively) to provide sufficient room for diphosphate egress, a requirement for water capture on the C3 re-face of the resulting carbocation and, conversely, implicating a role for this residue in promoting diphosphate capture at C3 during the required isomerization of GPP to LPP by the olefin cyclases. Furthermore, the relatively high substrate Km value noted for linalool synthase (more than fourfold higher than those of the olefin cyclases) is also consistent with the altered binding environment presented to the diphosphate by the displaced J/K loop with its resident aspartate residue.

B

C

Fig. 4. Models illustrating a portion of the J/K helices and the intervening J/K loops of linalool synthase (A), sabinene synthase (B), and limonene synthase (C) with geranyl diphosphate docked in the active sites as the left-handed helical conformer (A and B) or the right-handed helical conformer (C), consistent with the stereochemistry of the respective cyclization products. Models were generated using tobacco epi-aristolochene synthase [47] as the scaffold, with energy minimizations performed using GROMOS96 [48]. Geranyl diphosphate was docked manually using the relative positions of the conserved DDxxD motifs, divalent cations, and ligands in the structures of farnesyl diphosphate synthase (1UBW.pdb) and epi-aristolochene synthase (5EAT.pdb). The figure was produced using MOLSCRIPT [49].

Consistent with the implication of these models, limonene synthase mutant H579A yields an altered product profile with abundant monoterpenols (unpublished data), suggesting that replacing the steric bulk of the histidine with an alanine permits water access to intermediate carbocations in the same manner as displacement of the J/K loop removes the impediment to water access in linalool synthase. The altered position of the J/K loop in linalool synthase compared with the olefin cyclases provides a rationale for the observed differences between these enzyme types in diphosphate

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binding and water access to the active site. However, evaluation of the specific role(s) of this loop and its resident amino acids must await mutagenesis studies and crystal structure determination.

Acknowledgments This work was supported by NIH Grant GM-31354 and by Project 0268 from the Agricultural Research Center, Washington State University.

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