Functional analysis of (4S)-limonene synthase mutantsreveals determinants of catalytic outcome in a modelmonoterpene synthaseNarayanan Srividya, Edward M. Davis, Rodney B. Croteau1, and B. Markus Lange1

Institute of Biological Chemistry and M. J. Murdock Metabolomics Laboratory, Washington State University, Pullman, WA 99163

Contributed by Rodney B. Croteau, February 3, 2015 (sent for review November 8, 2014; reviewed by David E. Cane and Philip J. Proteau)

Crystal structural data for (4S)-limonene synthase [(4S)-LS] ofspearmint (Mentha spicata L.) were used to infer which amino acidresidues are in close proximity to the substrate and carbocationintermediates of the enzymatic reaction. Alanine-scanning muta-genesis of 48 amino acids combined with enzyme fidelity analysis[percentage of (−)-limonene produced] indicated which residuesare most likely to constitute the active site. Mutation of residuesW324 and H579 caused a significant drop in enzyme activity andformation of products (myrcene, linalool, and terpineol) character-istic of a premature termination of the reaction. A double mutant(W324A/H579A) had no detectable enzyme activity, indicating thateither substrate binding or the terminating reaction was impaired.Exchanges to other aromatic residues (W324H, W324F, W324Y,H579F, H579Y, and H579W) resulted in enzyme catalysts with sig-nificantly reduced activity. Sequence comparisons across theangiosperm lineage provided evidence that W324 is a conservedresidue, whereas the position equivalent to H579 is occupied byaromatic residues (H, F, or Y). These results are consistent with a crit-ical role of W324 and H579 in the stabilization of carbocation inter-mediates. The potential of these residues to serve as the catalyticbase facilitating the terminal deprotonation reaction is discussed.

monoterpene synthase | enzyme catalysis | mechanism | carbocation |structure–function relationship

Terpenoids are a structurally diverse group of metabolites withfunctions in both primary and secondary (or specialized)

metabolism. Primary metabolites derived from terpenoid path-way intermediates in plants include sterols, carotenoids, and theside chains of chlorophylls, tocopherols, and quinones of elec-tron transport systems. Many plant hormones are also products ofterpenoid metabolism, including abscisic acid, cytokinins, brassi-nosteroids, and strigolactones (1). Secondary plant metabolites ofterpenoid origin can play critical defense-related roles (e.g., ses-quiterpene lactones and triterpene saponins serve as antifeedants)and are dominant constituents of essential oils and resins (mono-,sesqui-, and diterpenes) (2). Terpene synthases (TPSs) converta prenyl diphosphate of a specific chain length to the first path-way-specific (often cyclic) intermediate in the biosynthesis of eachclass of terpenoids. Whereas some terpene synthases are re-markably specific and only generate one product from a prenyldiphosphate precursor, others release a larger number of productsfrom a common substrate, thus contributing to terpenoid chem-ical diversity (3). The genomes of plants may only contain oneTPS gene [e.g., ent-kaurene (diterpene) synthase in the mossPhyscomitrella patens (Hedw.) Bruch & Schimp.], but oftenharbor sizable families of TPS genes with more than 20 mem-bers, which is another source of terpenoid structural variety (4).All monoterpene synthases (MTSs) use either geranyl di-

phosphate (GPP) or its 2Z-isomer neryl diphosphate as sub-strate, but the sequence conservation across species is generallyfairly low (2). However, MTSs share a common tertiary structure(the so-called αβ fold), with a C-terminal α-domain containingthe active site and an N-terminal β-domain of as yet uncertain

function (3). The active site harbors a highly conserved, L-aspar-tate-rich, DDxxD motif, which is also found in prenyl elongases(5) and a less conserved NSE/DTE motif. The L-aspartate resi-dues bind a trinuclear cluster of divalent metal ions (Mg2+ orMn2+) involved in the binding and activation of the diphosphatemoiety, thereby generating characteristic carbocation intermediates(Fig. 1). Enzymes catalyzing these ionization-initiated cyclizationreactions are commonly referred to as class I TPSs (as opposed toclass II TPSs that initiate cyclizations by protonation) (3). Theremaining course of MTS catalysis is variable and generatesacyclic, monocyclic, and/or bicyclic monoterpenes.Because of the widespread occurrence of (−)-(4S)-limonene

throughout the plant kingdom, the relative simplicity of catalysis,the comparatively well-understood mechanism, and the avail-ability of a crystal structure at 2.7-Å resolution (6), (4S)-limo-nene synthase [(4S)-LS] has become a model for understandingcatalysis by class I TPSs. The (4S)-LS gene is translated intoa preprotein with an N-terminal targeting sequence for trans-port to the plastidial envelope membrane (7). Based on resultsobtained with a series of truncated (4S)-LS mutants, the mostlikely cleavage site of the preprotein was determined to be at (ornear) a tandem pair of arginines (R58 and R59 of the preprotein)(8). If truncated beyond R58, (4S)-LS shows no cyclization ac-tivity with GPP but is fully functional with linalyl diphosphate(LPP) as a substrate. Interestingly, the crystal structure of (4S)-LS,complexed with the nonhydrolyzable LPP analog 2-fluorolinalyldiphosphate (FLPP), does not provide evidence for a directinteraction of R58 or R59 with the substrate (9). However, weak

Significance

Terpene synthases catalyze complex, chain length-specific, elec-trophilic cyclization reactions that constitute the first committedstep in the biosynthesis of structurally diverse terpenoids.(4S)-limonene synthase [(4S)-LS] has emerged as a model en-zyme for enhancing our comprehension of the reaction cycleof monoterpene (C10) synthases. While the stereochemistryof the cyclization of geranyl diphosphate to (−)-(4S)-limonenehas been the subject of several mechanistic studies, the struc-tural basis for the stabilization of carbocation intermediates andthe termination of the reaction sequence have remained enig-matic. We present extensive experimental evidence that the ar-omatic amino acids W324 and H579 play critical roles in thestabilization of intermediate carbocations. A possible function ofthese residues as the terminal catalytic base is also discussed.

Author contributions: N.S., E.M.D., R.B.C., and B.M.L. designed research; N.S. and E.M.D.performed research; N.S., R.B.C., and B.M.L. analyzed data; and N.S. and B.M.L. wrote thepaper.

Reviewers: D.E.C., Brown University; and P.J.P., Oregon State University.

The authors declare no conflict of interest.1To whom correspondence may be addressed. Email: croteau@wsu.edu or lange-m@wsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1501203112/-/DCSupplemental.

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interactions, in particular electrostatic interplay of R58 withE363 and hydrogen bonding between R59 and V357/Y435, ap-pear to anchor the N-terminal strand to the outside of the activesite, thereby possibly supporting the closure of the active site,while not interfering with the binding of the substrate andintermediates (9). The reaction mechanism of (4S)-LS fromspearmint (Mentha spicata L.) has been studied in some detail.The catalytic cascade involves the migration of the diphosphategroup to C3 of the geranyl cation (from the original C1) to affordenzyme-bound (3S)-LPP as an intermediate (10) (Fig. 1). Fol-lowing C2–C3 rotation, the diphosphate is released again togenerate a linalyl cation. The proximity of the C6–C7 doublebond to the positive charge facilitates an anti-SN′ cyclization toform the (−)-(4S)-terpinyl cation (11, 12) (Fig. 1). Deprotona-tion from the adjacent methyl group (C8 of the original GPP)yields the monocyclic olefin (−)-(4S)-limonene as the majorproduct (96%). Side products are obtained by either prematuredeprotonation of the geranyl cation to generate the acyclic olefinmyrcene (2%) or additional cyclization of the terpinyl cation(between C2 and C7 of the original GPP) to produce, afterdeprotonation, a mixture of the bicyclic olefins α- and β-pinene(2%) (10, 13) (Fig. 1).Using synthetic analogs of GPP with a chiral methyl group at

C9 (carrying 1H, 2H, and 3H), Coates et al. demonstrated thatthe final deprotonation occurs predominantly by re-facial anti-elimination (12). It has been hypothesized that the diphosphateanion released from the prenyl diphosphate substrate may act as

the catalytic base for this deprotonation in various prenyl di-phosphate synthases and terpene synthases, but no direct evi-dence is available to date (3, 14–17) and it seems highly unlikelyin the present case due to spatial considerations. Here we pres-ent a comprehensive dataset to map the active site of spearmint(4S)-LS and evaluate residues with potential roles in stabilizingcarbocation intermediates of the reaction cycle. We also discussthe broader implications of our findings for understanding catal-ysis and reaction termination by this fascinating class of enzymes.

ResultsL-Alanine Scanning Mutagenesis Defines Residues Required forSubstrate Binding and/or Catalysis. X-ray data for the crystal struc-ture of the pseudomature form of spearmint (4S)-LS (R58; lack-ing the plastidial targeting sequence), complexed with FLPP asa nonhydrolyzable analog of the reaction intermediate LPP (9),were used to infer the amino acid residues within closest prox-imity to the substrate (Dataset S1). Distances were calculatedbetween each of the 10 carbon atoms of the substrate analog andthe carbon atoms of all amino acids of the protein (except forthose forming peptide bonds). The 48 residues located closest tothe substrate analog were exchanged for L-alanine by site-directedmutagenesis (introduced into the pseudomature form of (4S)-LSthat is truncated at R58), and these mutant enzymes were ex-pressed in Escherichia coli. Following purification over hy-droxyapatite, each recombinant enzyme was incubated with GPP

MONOCYCLIC HYDROCARBON

BICYCLIC HYDROCARBONS

MONOCYCLIC ALCOHOLSBICYCLIC ETHER

ACYCLIC HYDROCARBON

ACYCLIC ALCOHOLS

Fig. 1. Proposed mechanism for (4S)-limonene synthase catalysis. OPP denotes the diphosphate moiety. The primary pathway in the wild-type enzyme leadsto the formation of (−)-limonene (dark gray) and smaller amounts of bicyclic and acyclic products (light gray). Other products shown in this figure are releasedby mutant enzymes.

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and products of the reaction were analyzed by quantitative chiralphase gas chromatography.Mutations affecting the conserved L-aspartate residues known

to be of crucial importance for complexing divalent active sitemetal ions (Mg2+ or Mn2+) in monoterpene synthases and re-lated enzymes (5, 18), D352A, D353A, and D496A, predictablyresulted in inactive enzymes (Dataset S2). The majority of theremaining mutants generated product profiles similar to those ofthe wild-type enzyme [(−)-limonene (96.6%), myrcene (1.0%),(−)-α-pinene (0.6%), (+)-α-pinene (0.7%), (−)-β-pinene (0.5%),(+)-β-pinene (0.5%), and (+)-sabinene (0.3%)]. However, somesingle residue exchanges led to a significantly reduced rateof (−)-limonene production, with increases in the formationof side products and novel monoterpenes not detectable in assayswith the wild-type enzyme (Dataset S2). The acyclic monoter-pene myrcene was produced in substantial amounts by M458A(10.1%), I348A (9.7%), and T349A (8.3%). Acyclic monoterpenealcohols were released at fairly high levels from mutant enzymesW324A [equal amounts (26.7% each) of (−)- and (+)-linalool],M458A [equal amounts (14.3% each) of (−)-and (+)-linalool],and H579A [8.6% (−)-linalool] (Dataset S2). A novel productderived from an altered reaction stereochemistry was (4R)-(+)-limonene generated by M458A (8.6%). Monocyclic alcoholswere found to be novel products of M458A [27.3% (+)-α-terpineol],H579A [25.0% (−)-α-terpineol], and L492A [8.0% (-)-α-terpineol](Dataset S2). Increased amounts of bicyclic products were formedby N345A [36.6% (−)-sabinene, 14.8% (+)-α-pinene, and 13.9%(+)-β-pinene]. Additional novel products generated in smalleramounts by mutant enzymes were α-thujene (N345A) and1,8-cineole (T349A, L492A, and M458A) (Dataset S2).To further assess the observed loss in fidelity in mutant

enzymes, we plotted the distance between each amino acid andthe substrate analog in the (4S)-LS crystal structure against thepercentage of (−)-limonene released by the enzyme after thisresidue was mutated to L-alanine (Fig. 2). Interestingly, muta-tions affecting the nine amino acid residues positioned in closestproximity to the substrate analog (i.e., those with a carbon atomat a distance of less than 5 Å to a carbon atom of the substrateanalog) released (−)-limonene at less than 80% of the products,indicating a possible direct role in substrate (or intermediate)binding and/or catalysis. The only exceptions were D352A andD353A (with minimum C–C distances of 5.1 and 8.1 Å, re-spectively), which are known to be required for the divalent metalion-mediated binding of the pyrophosphate after its release from

the prenyl diphosphate, and thus do not interact with the prenylor emerging terpenoid moiety (3) (Fig. 2).

W324A and H579A Mutants Are Catalytically Impaired and PreferentiallyGenerate an Acyclic Hydrocarbon and Monoterpene Alcohols. Underroutine end-point assay conditions (200 μg purified protein), allavailable substrate (GPP concentration at 5 mM) was turned overby the wild-type and most mutant enzymes. However, two mutants,W324A and H579A, converted only small amounts of substrateduring the 2-h time frame of the assay. Under kinetic assay con-ditions, when the R58 wild-type enzyme converts ≤50% of thesubstrate, the specific activity of R58 was 0.19 nmol (h × mg pro-tein)−1, and the catalytic constants determined in our assays weresimilar to those reported in the literature (Km = 12.6 μM; kcat =0.037 s−1) (8). The specific activities of the W324A and H579Amutant enzymes were significantly reduced and, therefore, kineticvalues could not be determined with accuracy.To further investigate the potential roles of W324 and H579 in

(4S)-LS catalysis, additional mutants were generated. W324 andH579 were exchanged with another aromatic residue (W324H,W324F, W324Y, H579F, H579Y, and H579W), a base (W324H,W324K, and H579K), or a randomly selected amino acid. Themajority of W3234 mutant enzymes had extremely low specificactivities [≤0.02 nmol (h × mg protein)−1; 3–10% of wild type],the only exception being W324F and W324Y [0.08 and 0.06 nmol(h × mg protein)−1; 50% and 32% of wild-type activity, re-spectively]. The H579 mutants had specific activities rangingbetween 40% and 50% of those of the wild-type enzyme. Theproduct profiles were significantly altered in all 12 W324 mutants(W324A, W324C, W324F, W324H, W324I, W324K, W324L,W324P, W324Q, W324S, W324T, and W324Y) and five of nineH579 mutants (H579A, H579D, H579K, H579N, and H579W)(Dataset S3). The majority of W324 and H579 mutants gen-erated notable amounts of the acyclic hydrocarbon myrcene(Fig. 3), which is released by premature deprotonation from thegeranyl or linalyl cation (Fig. 1). The most prominent products ofW324 mutants were acyclic alcohols, the formation of which iscaused by premature quenching of an early carbocation in-termediate (linalyl cation) by a water molecule (Fig. 1). Anexample is W324P, which forms 39.6% (−)-linalool and 40.5%(+)-linalool (80.1% acyclic alcohols) (Fig. 3A). Another notable classof products was monocyclic alcohols [(−)-α- and (+)-α-terpineol;detectable in 11 W324 mutants], which are generated by quenchingof the terpinyl cation by a water molecule (Fig. 1) (19). The highestrelative amounts were produced by W324A [11.6% (−)-α-terpineoland 5.7% (+)-α-terpineol; 17.3% moncyclic alcohols] (Fig. 3A). Anexception was the W324H mutant which, in addition to mono-terpene alcohols, produced high amounts of bicyclic monoterpenes[42.0% (+)-sabinene and 7.8% (−)-sabinene]. The catalytically im-paired H579 mutants, in agreement with W324 mutant data, werealso characterized by the production of considerable amounts ofacyclic and monocyclic alcohols. A representative example isH579K, which released 14.9% (−)-linalool, 14.9% (+)-linalool,22.1% (−)-α-terpineol, and 4.1% (+)-α-terpineol (56.0% alco-hols) (Fig. 3B). A W324A/H579A double mutant had no de-tectable enzyme activity.

Discussion(4S)-LS Mutants with Substituted Active-Site Residues Lose Fidelity.An analysis of the X-ray crystallographic structure of (4S)-LS (8),complexed with a substrate analog, enabled us to hypothesizewhich amino acid residues are likely to form the active site of theenzyme. We further hypothesized that exchanges of residuesinvolved in substrate binding and catalysis would generate mu-tant enzymes with lower specific activity and/or result in alteredproduct profiles. A total of 48 amino acid residues were found tobe arranged within a radius of about 25 Å from any carbon atomof the C10 substrate analog. When these residues were substituted

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D496AD352AD353A

noitamrof

enenomiL

latotfo%[

senepretonom

]

Closest C-C distance to substrate [Å]

Fig. 2. Mutant enzymes lose fidelity when residues that likely function insubstrate binding and/or catalysis are exchanged. The closest distance ofa carbon atom of each amino acid in (4S)-limonene synthase is plottedagainst the percentage of (−)-limonene formation when the residue is ex-changed with L-alanine.

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with L-alanine, the mutant enzymes were mostly unaffected intheir specific activity and fidelity. However, 11 mutants released(−)-limonene as less than 80% of the total monoterpene products.In these fidelity-impaired enzymes, a residue containing a carbonatom within close proximity to a substrate carbon atom was mu-tated (Fig. 4A). Strong hydrogen bonding below 3 Å can thusoccur between donor oxygens or nitrogens of active site aminoacids (e.g., –OH in T349, or S454; –NH– in W324 or H579) andacceptor sites of the substrate or intermediates. By analogy to anearlier mechanistic proposal for 5-epi-aristolochene synthase(TEAS) (20), we propose that the carbocation reaction inter-mediates of (4S)-LS might move deeper into a hydrophobicpocket [composed of W324, N345, T349, S454, M458, and H579in (4S)-LS], which would enhance the stabilization of the positivecharge by aromatic residues situated at the bottom of the activesite. The region of positive charge around the divalent metal ionsinteracts directly with the diphosphate leaving group, therebyimmobilizing this anion and preventing a recapture of the allyliccarbocation (which would regenerate GPP and terminate thereaction) (20, 21).

W324 and H579 Appear to Play Roles in Stabilizing the α-TerpinylCation. Following the cyclization step, the positive charge of theα-terpinyl cation could be stabilized by cation-π interactions withthe electron-rich heteronuclear aromatic rings of W324 andH579, which would be particularly strong if the carbocation weresituated, as hypothesized, in the hydrophobic active site pocket(Fig. 4B). Analogous stabilizing roles had been proposed for aro-matic residues in sesquiterpene synthases (20, 21). The importance

of L-histidine in TPS catalysis was already recognized by Rajao-narivony et al. (22) based on experiments with histidine-directedinhibitors, but the data presented here provide to our knowledgethe first evidence for the involvement of a specific residue (H579).One would predict that, if a charge-stabilizing residue was ex-changed with a nonaromatic residue, the reaction outcome wouldreflect early termination products. Indeed, almost all W324 mutantsreleased significant amounts of acyclic myrcene and (+/−)-linalool(up to 83% of total products), which is indicative of a reaction thatterminates before the cyclization to the α-terpinyl cation (Fig. 1).Interestingly, W324F and W324Y also formed mostly early termi-nation products (77% and 81%, respectively), indicating that otheraromatic amino acids could not effectively substitute for W324.It is thus not surprising that the W324 residue is conserved in an-giosperm MTSs (Dataset S4). H579 mutants generally releaseda mixture of myrcene, (+/−)-linalool and (+/−)-α-terpineol,with the exception of H579F and H579Y (aromatic substitutions),which, similar to the wild-type enzyme, generated (−)-limonene asthe only major product (>85%). Interestingly, angiosperm MTSsthat generate cyclized products carry an aromatic residue (H, F, orY) in the position corresponding to H579 in (4S)-LS. The for-mation of cyclic alcohols by W324 and H579 mutants providesevidence that these residues are also required for avoidingquenching of the reaction by water, thereby further stabilizingthe α-terpinyl cation for subsequent deprotonation. The high-resolution structure of (+)-bornyl diphosphate synthase (at 2.0 Å)indicated the presence of water molecules in the active site of thisMTS (19), which could not be discerned in the lower resolutionstructure (at 2.7 Å) of (4S)-LS (8). (+/−)-Linalool (formed byW324 mutants) would be expected to be generated by watercapture of the linalyl cation, where the charge is closer to themetal ion-bound pyrophosphate at the top of the active site.(+/−)-α-Terpineol (formed in H579 mutants) would be predictedto be produced by reaction with water approaching from thebottom of the active site. It is presently unknown whether watermolecules are admitted to the active site in W324 and H579mutants or, if already present in wild-type (4S)-LS, are permittedto react with carbocation intermediates in these mutants.

Which Residues Are Involved in the Final Deprotonation Reaction?The ultimate steps of the (4S)-LS reaction cascade are thedeprotonation of a carbocation and release of the olefin finalproduct. Several authors have previously proposed that the ini-tially released pyrophosphate may act as the catalytic base (3,14–17). This is likely correct for bornyl diphosphate synthase(BPPS) from culinary sage (Salvia officinalis L.), where the py-rophosphate is recaptured in the terminating step of the reaction(19, 23). However, such a recapture does not occur in othercharacterized MTSs and, if the carbocation was situated andstabilized, as hypothesized, in a hydrophobic pocket of the activesite, then the metal ion-bound diphosphate would be too distantto act as a base (Fig. 4). Based on quantum mechanical calcu-lations, Hong and Tantillo (24) proposed pathways for the for-mation of BPP by BPPS. In all models, the bicyclic bornyl cationassumes a conformation that positions C2 for a recapture of thediphosphate anion, and we propose that, in other MTSs, theα-terpinyl carbocation interacts with the charge-stabilizing aro-matic residues W324 (conserved) and H579 (or other aromaticresidues in the same position) at the bottom of the active sitecavity (separated from the diphosphate anion, which is bound byresidues forming the top of the active site).H579 has the properties of a catalytic base and, in one energy-

minimized docking orientation of the α-terpinyl cation, is posi-tioned close to the proton to be removed (Fig. 4C). W324 issituated close to the leaving proton in an alternative dockingorientation of the α-terpinyl cation (Fig. 4D). Whereas H579could be the primary base in wild-type (4S)-LS, it can be ex-changed with other, nonbasic, residues with only minor effects on

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Fig. 3. Mutant enzymes produce acyclic monoterpene olefins, and acyclicand monocyclic monoterpene alcohols, when amino acid exchanges aremade in positions occupied by residues required for stabilizing the α-terpinylcation. (A) W324 and (B) H579 mutants.

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enzyme activity. If W324 were to act as a catalytic base (resultingin a protonated indole with an approximate pKa of −2) (25), theprotonation would occur preferentially at Cγ, which is a possi-bility because the α-terpinyl cation is a strong conjugate acid(pKa ∼ −10). The significant reduction of specific enzyme ac-tivity in all W324 mutants could be interpreted as evidence that thetermination reaction (rather than the initial isomerization) (10) hasbecome rate limiting. The fact that the W324A/H579A doublemutant had no measurable enzyme activity is consistent with afunction of these residues as potential catalytic bases. An alterna-tive explanation would be that W324 (and even more so W324/H579) mutations cause an aberrant binding of GPP. This issue canonly be resolved with a series of more detailed kinetic evaluationsoutside the scope of the current study. The work presented hereconstitutes the most extensive combination of mutagenesis andproduct analysis in a model MTS, and it can be concluded that verysubtle modifications in active site structure can result not only inalteration of termination chemistry, but also in the regiochemistryand stereochemistry of the cyclization reaction itself. The native(4S)-LS, an enzyme of very high fidelity, must therefore guide thereaction course very precisely to avoid such digression. Our dataare the foundation for future efforts to better understand the as yetunpredictable outcome of reactions catalyzed by MTSs.

Materials and MethodsCalculating C–C Distances from (4S)-LS Crystal Structure. Coordinates of thecarbon atoms of all amino acids were obtained from the published crystalstructure of (4S)-LS (PDB accession identifier 2ONH; complexed with the C10

substrate analog FLPP). A perl script was used to calculate each distanceusing the following formula:

DðC1 −C2Þ=ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðx1 − x2Þ2 + ðy1 − y2Þ2 + ðz1 − z2Þ2

q:

Site-Directed Mutagenesis. The spearmint (4S)-LS cDNA, truncated at R58 toeliminate the plastidial targeting sequence, was cloned into the pSBETvector (26) as described before (8). Point mutations were introduced by PCRusing a modified overlap extension strategy as outlined previously (27). Acomplete list of primers used in these reactions is provided in Dataset S5. PCRamplicons were digested with BamHI and NdeI, gel purified, and ligated intoa similarly digested and purified vector. The products of the ligation reac-tions were transformed into XL-1 blue competent cells and plated onto LB-agar plates containing 50 μg/mL kanamycin. Individual colonies were pickedand bacteria grown under selection pressure. Plasmid DNA was then isolatedusing a commercial kit (GeneJet Mini Prep kit, Fermentas–Thermo FisherScientific) and the inserted gene sequence was confirmed by a commercialservice provider (Eurofins Genomics).

Recombinant Protein Expression and Purification. Vectors containing (4S)-LSwild-type (R58) and mutant genes were transformed into E. coli BL21 DE3cells, which were then grown overnight to an OD of ∼1.0 at 37 °C in LBmedium containing 50 μg/mL kanamycin. The cultures were induced with0.5 mM isopropyl-1-thio-β-D-galactopyranoside and grown for another 24 hat 16 °C. Cells were harvested by centrifugation at 2,500 × g for 10 min, sus-pended in a cell disruption buffer [50 mM MOPSO, 10 mM DTT, 10% (vol/vol)glycerol; pH 7.0], and sonicated on ice three times for 15 s each. The su-pernatant (400 μL) obtained after centrifugation at 15,000 × g was mixedwith 100 mg hydroxyapatite [preequilibrated with 10 mM sodium phos-phate (pH 7.0)] in an Eppendorf tube and gently mixed by tube inversion for1 h at 4 °C. The hydroxyapatite was then allowed to settle by gravity and thesupernatant was removed with a Pasteur pipette. Weakly bound proteinswere removed by washing with 50 mM MOPSO (pH 7.0). (4S)-LS was eluted

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Fig. 4. Possible roles of W324 and H579. The carbon skeletons of the sub-strate analog (FLPP) and the α-terpinyl cation intermediate are depicted in

green. (A) Residues lining the active site of (4S)-limonene synthase witha bound substrate analog based on crystal structural data (9). (B) Proposedstabilization of the α-terpinyl cation intermediate by carbocation-π inter-actions with H579 and/or W324. (C) Orientation of α-terpinyl cation so thatH579 can act as a catalytic base. (D) Orientation of α-terpinyl cation so thatW324 can act as a catalytic base. Note that the deprotonation reactions inC and D would both result in a product with the correct stereochemistry[(−)-limonene].

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with 100 mM sodium phosphate (pH 7.0). SDS gel electrophoresis indicateda >90% purity of the recombinant enzymes.

Enzyme Assays. Purified enzyme (200 μg) was reacted with 0.5 mM GPP(obtained synthetically according to ref. 28) in 400 μL MOPSO buffer (pH 7.0)overlayed with 100 μL hexane for 16 h at 31 °C (gentle agitation). Enzymaticreactions were terminated by vigorous mixing of the aqueous and organicphases, and phase separation was achieved by placing samples in a freezer(−20 °C) for 2 h. The upper hexane layer was removed for analysis by gaschromatography with flame ionization detection (model 7890, AgilentTechnologies) under the following conditions: injector at 250 °C, 20:1 splitinjection mode (1 μL); detector at 250 °C (H2 flow at 30 mL/min, airflow at400 mL/min, makeup flow (He) at 25 mL/min); Cyclodex-B chiral column(J&W Scientific 112–2532; 30 m × 0.25 mm, 0.25 μm film thickness)operated at 2 mL/min flow rate with He as carrier gas; oven heatingprogram starting at 40 °C with a ramp to 120 °C at 2°/min, a second rampto 200 °C at 50 °C/min, and a final hold at 200 °C for 2 min. Peaks wereidentified based on comparisons of retention indices with those of au-thentic standards and verified where necessary by standard GC-MSmethods (29). For kinetic assays, enzymatic assay times were adjusted to2 h to ensure that no more than 20% of the available substrate was

consumed. Kinetic parameters were determined by varying substrateconcentrations while maintaining other reactants at saturation. Kineticconstants (Km and Kcat) were calculated by nonlinear regression analysis(Origin 8; OriginLab).

Carbocation Structure and Docking. The molecular geometry of the α-terpinylcation was optimized by ab initio electronic structure calculation and mini-mization (using default values for bond length, bond angles, and dihedralangles) with the Gaussian03 program at the HF/6–31G level (Gaussian Inc.).The optimized geometry output file was converted into the standard formatfor the Protein Data Bank. Molecular docking of the α-terpinyl cation in the(4S)-LS active site was performed with AutoDock Vina (vina.scripps.edu/)using default settings.

ACKNOWLEDGMENTS. The authors thank Ms. Lisa Washburn and Ms.Deanna Heidorn for technical assistance. This work was supported by theDivision of Chemical Sciences, Geosciences, and Biosciences, Office of BasicEnergy Sciences, US Department of Energy Grant DE-FG02-09ER16054 (toB.M.L.) for the biochemical characterization of mutant enzymes and theNational Institutes of Health Grant GM-31354 (to R.B.C.) for the genera-tion of site-directed mutants.

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