Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase Narayanan 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] of function (3). The active site harbors a highly conserved, L-aspar- spearmint (Mentha spicata L.) were used to infer which amino acid tate-rich, DDxxD motif, which is also found in prenyl elongases residues are in close proximity to the substrate and carbocation (5) and a less conserved NSE/DTE motif. The L-aspartate resi- + intermediates of the enzymatic reaction. Alanine-scanning muta- dues bind a trinuclear cluster of divalent metal ions (Mg2 or + genesis of 48 amino acids combined with enzyme fidelity analysis Mn2 ) involved in the binding and activation of the diphosphate [percentage of (−)-limonene produced] indicated which residues moiety, thereby generating characteristic carbocation intermediates are most likely to constitute the active site. Mutation of residues (Fig. 1). Enzymes catalyzing these ionization-initiated cyclization W324 and H579 caused a significant drop in enzyme activity and reactions are commonly referred to as class I TPSs (as opposed to formation of products (myrcene, linalool, and terpineol) character- class II TPSs that initiate cyclizations by protonation) (3). The istic of a premature termination of the reaction. A double mutant remaining course of MTS catalysis is variable and generates (W324A/H579A) had no detectable enzyme activity, indicating that acyclic, monocyclic, and/or bicyclic monoterpenes. either substrate binding or the terminating reaction was impaired. Because of the widespread occurrence of (−)-(4S)-limonene Exchanges to other aromatic residues (W324H, W324F, W324Y, throughout the plant kingdom, the relative simplicity of catalysis, H579F, H579Y, and H579W) resulted in enzyme catalysts with sig- the comparatively well-understood mechanism, and the avail- nificantly reduced activity. Sequence comparisons across the ability of a crystal structure at 2.7-Å resolution (6), (4S)-limo- angiosperm lineage provided evidence that W324 is a conserved nene synthase [(4S)-LS] has become a model for understanding residue, whereas the position equivalent to H579 is occupied by catalysis by class I TPSs. The (4S)-LS gene is translated into aromaticresidues(H,F,orY).Theseresultsareconsistentwithacrit- a preprotein with an N-terminal targeting sequence for trans- ical role of W324 and H579 in the stabilization of carbocation inter- port to the plastidial envelope membrane (7). Based on results mediates. The potential of these residues to serve as the catalytic obtained with a series of truncated (4S)-LS mutants, the most base facilitating the terminal deprotonation reaction is discussed. likely cleavage site of the preprotein was determined to be at (or near) a tandem pair of arginines (R58 and R59 of the preprotein) monoterpene synthase | enzyme catalysis | mechanism | carbocation | (8). If truncated beyond R58, (4S)-LS shows no cyclization ac- structure–function relationship tivity with GPP but is fully functional with linalyl diphosphate (LPP) as a substrate. Interestingly, the crystal structure of (4S)-LS, erpenoids are a structurally diverse group of metabolites with complexed with the nonhydrolyzable LPP analog 2-fluorolinalyl Tfunctions in both primary and secondary (or specialized) diphosphate (FLPP), does not provide evidence for a direct metabolism. Primary metabolites derived from terpenoid path- interaction of R58 or R59 with the substrate (9). However, weak way intermediates in plants include sterols, carotenoids, and the side chains of chlorophylls, tocopherols, and quinones of elec- Significance tron transport systems. Many plant hormones are also products of terpenoid metabolism, including abscisic acid, cytokinins, brassi- Terpene synthases catalyze complex, chain length-specific, elec- nosteroids, and strigolactones (1). Secondary plant metabolites of trophilic cyclization reactions that constitute the first committed terpenoid origin can play critical defense-related roles (e.g., ses- step in the biosynthesis of structurally diverse terpenoids. quiterpene lactones and triterpene saponins serve as antifeedants) (4S)-limonene synthase [(4S)-LS] has emerged as a model en- and are dominant constituents of essential oils and resins (mono-, zyme for enhancing our comprehension of the reaction cycle sesqui-, and diterpenes) (2). Terpene synthases (TPSs) convert of monoterpene (C10) synthases. While the stereochemistry a prenyl diphosphate of a specific chain length to the first path- of the cyclization of geranyl diphosphate to (−)-(4S)-limonene way-specific (often cyclic) intermediate in the biosynthesis of each has been the subject of several mechanistic studies, the struc- class of terpenoids. Whereas some terpene synthases are re- tural basis for the stabilization of carbocation intermediates and markably specific and only generate one product from a prenyl the termination of the reaction sequence have remained enig- diphosphate precursor, others release a larger number of products matic. We present extensive experimental evidence that the ar- from a common substrate, thus contributing to terpenoid chem- omatic amino acids W324 and H579 play critical roles in the ical diversity (3). The genomes of plants may only contain one stabilization of intermediate carbocations. A possible function of TPS gene [e.g., ent-kaurene (diterpene) synthase in the moss theseresiduesastheterminalcatalyticbaseisalsodiscussed. Physcomitrella patens (Hedw.) Bruch & Schimp.], but often harbor sizable families of TPS genes with more than 20 mem- 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 the bers, which is another source of terpenoid structural variety (4). paper. All monoterpene synthases (MTSs) use either geranyl di- Reviewers: D.E.C., Brown University; and P.J.P., Oregon State University. Z phosphate (GPP) or its 2 -isomer neryl diphosphate as sub- The authors declare no conflict of interest. strate, but the sequence conservation across species is generally 1To whom correspondence may be addressed. Email: [email protected] or lange-m@wsu. fairly low (2). However, MTSs share a common tertiary structure edu. αβ α (the so-called fold), with a C-terminal -domain containing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the active site and an N-terminal β-domain of as yet uncertain 1073/pnas.1501203112/-/DCSupplemental. 3332–3337 | PNAS | March 17, 2015 | vol. 112 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1501203112 Downloaded by guest on September 29, 2021 ACYCLIC ACYCLIC ALCOHOLS HYDROCARBON BICYCLIC HYDROCARBONS MONOCYCLIC HYDROCARBON MONOCYCLIC ALCOHOLS BICYCLIC ETHER Fig. 1. Proposed mechanism for (4S)-limonene synthase catalysis. OPP denotes the diphosphate moiety. The primary pathway in the wild-type enzyme leads to the formation of (−)-limonene (dark gray) and smaller amounts of bicyclic and acyclic products (light gray). Other products shown in this figure are released by mutant enzymes. interactions, in particular electrostatic interplay of R58 with the catalytic base for this deprotonation in various prenyl di- E363 and hydrogen bonding between R59 and V357/Y435, ap- phosphate synthases and terpene synthases, but no direct evi- pear to anchor the N-terminal strand to the outside of the active dence is available to date (3, 14–17) and it seems highly unlikely BIOCHEMISTRY site, thereby possibly supporting the closure of the active site, in the present case due to spatial considerations. Here we pres- while not interfering with the binding of the substrate and ent a comprehensive dataset to map the active site of spearmint intermediates (9). The reaction mechanism of (4S)-LS from (4S)-LS and evaluate residues with potential roles in stabilizing Mentha spicata spearmint ( L.) has been studied in some detail. carbocation intermediates of the reaction cycle. We also discuss The catalytic cascade involves the migration of the diphosphate the broader implications of our findings for understanding catal- group to C3 of the geranyl cation (from the original C1) to afford ysis and reaction termination by this fascinating class of enzymes. enzyme-bound (3S)-LPP as an intermediate (10) (Fig. 1). Fol- lowing C2–C3 rotation, the diphosphate is released again to Results – generate a linalyl cation. The proximity of the C6 C7 double L-Alanine Scanning Mutagenesis Defines Residues Required for bond to the positive charge facilitates an anti-SN′ cyclization to − S Substrate Binding and/or Catalysis. X-ray data for the crystal struc- form the ( )-(4 )-terpinyl cation (11, 12) (Fig. 1). Deprotona- ture of the pseudomature form of spearmint (4S)-LS (R58; lack- tion from the adjacent methyl group (C8 of the original GPP) ing the plastidial targeting sequence), complexed with FLPP as yields the monocyclic olefin (−)-(4S)-limonene as the major a nonhydrolyzable analog of the reaction intermediate LPP (9), product (96%). Side products are obtained by either premature deprotonation of the geranyl cation to generate the acyclic