Plant Terpenoid Synthases: Molecular Biology and Phylogenetic Analysis (Terpene Cyclase͞isoprenoids͞plant Defense͞genetic Engineering͞secondary Metabolism)
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 4126–4133, April 1998 Biochemistry This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 1997. Plant terpenoid synthases: Molecular biology and phylogenetic analysis (terpene cyclaseyisoprenoidsyplant defenseygenetic engineeringysecondary metabolism) JO¨RG BOHLMANN*†,GILBERT MEYER-GAUEN‡, AND RODNEY CROTEAU*§ *Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340; and ‡Human Genetics Center, University of Texas, Houston, TX 77225 Contributed by Rodney Croteau, February 25, 1998 ABSTRACT This review focuses on the monoterpene, field is periodically surveyed (10, 11). After brief coverage of the sesquiterpene, and diterpene synthases of plant origin that use three types of terpene synthases from higher plants, with empha- the corresponding C10,C15, and C20 prenyl diphosphates as sis on common features of structure and function, we focus here substrates to generate the enormous diversity of carbon on molecular cloning and sequence analysis of these important skeletons characteristic of the terpenoid family of natural and fascinating catalysts. products. A description of the enzymology and mechanism of Enzymology and Mechanism of Terpenoid Cyclization terpenoid cyclization is followed by a discussion of molecular cloning and heterologous expression of terpenoid synthases. GDP is considered to be the natural substrate for monoterpene Sequence relatedness and phylogenetic reconstruction, based synthases, because all enzymes of this class efficiently utilize this on 33 members of the Tps gene family, are delineated, and precursor without the formation of free intermediates (12). Since comparison of important structural features of these enzymes GDP cannot be cyclized directly because of the C2-C3 trans- is provided. The review concludes with an overview of the double bond, the reaction mechanism necessarily involves both organization and regulation of terpenoid metabolism, and of isomerization and cyclization steps (12, 13). Thus, GDP ionizes the biotechnological applications of terpenoid synthase genes. with the assistance of a divalent metal ion (Fig. 2), as in the first step of the prenyltransferase reaction (8). The resulting allylic The pathways of monoterpene, sesquiterpene, and diterpene cation-diphosphate anion pair then rearranges to form the en- biosynthesis are conveniently divided into several stages. The first zyme-bound, tertiary allylic isomer, 3R-or3S-linalyl diphosphate encompasses the synthesis of isopentenyl diphosphate, isomer- (LDP, depending on the initial folding of the geranyl substrate). ization to dimethylallyl diphosphate, prenyltransferase-catalyzed After rotation to the cisoid conformer, LDP ionizes and is cyclized in anti,endo-form to the corresponding 4R-or4S-a- condensation of these two C5-units to geranyl diphosphate (GDP), and the subsequent 19-4 additions of isopentenyl diphos- terpinyl cation. From this universal intermediate, the reaction may take one of several routes involving internal additions to the phate to generate farnesyl (FDP) and geranylgeranyl (GGDP) remaining double bond, hydride shifts, or rearrangements before diphosphate (Fig. 1) (1). In the second stage, the prenyl diphos- the terminal carbocation is deprotonated to an olefin or captured phates undergo a range of cyclizations based on variations on the by water or the diphosphate anion. In the simplest of all terpenoid same mechanistic theme to produce the parent skeletons of each cyclizations, the a-terpinyl cation is deprotonated to yield li- class. Thus, GDP (C ) gives rise to monoterpenes (2), FDP (C ) 10 15 monene (14) (Fig. 2). Alternatively, the a-terpinyl cation may to sesquiterpenes (3), and GGDP (C ) to diterpenes (4). These 20 undergo further cyclization, via the remaining double bond, to transformations catalyzed by the terpenoid synthases (cyclases) afford the pinyl cation (and then a-orb-pinene after deproto- may be followed by a variety of redox modifications of the parent nation) (15) or the bornyl cation (to form bornyl diphosphate by skeletal types to produce the many thousands of different terpe- capture of the diphosphate) (16). Hydride shifts in the a-terpinyl noid metabolites of the essential oils, turpentines, and resins of cation yield the terpinen-3-yl or terpinen-4-yl cations, providing plant origin (5). access to the phellandrenes (17) and thujanes (18), respectively. This review focuses on the synthases that use prenyl diphos- Water capture of the a-terpinyl cation yields a-terpineol, which phate substrates to generate the enormous diversity of carbon upon heterocyclization affords 1,8-cineole (19). All monoterpene skeletons characteristic of terpenoids. Most of these natural cyclases are capable of catalyzing both the isomerization and products are cyclic, and many contain multiple ring systems, the cyclization reactions, and these steps occur via a series of ion pairs basic structures of which are determined by the highly specific at the same active site (12). A few monoterpene synthases terpenoid synthases; examples of synthases that produce acyclic produce acyclic products such as myrcene (20) and linalool (21). products are also known. The terpenoid synthases may be in- Additional variations on the electrophilic isomerization– volved in the regulation of pathway flux because they operate at cyclization sequences illustrated (Fig. 2) account for essentially all metabolic branch points and catalyze the first committed steps monoterpene skeletal types, and their stereoisomers and deriv- leading to the various terpene classes (6). The synthases respon- atives. sible for generating the parent compounds of the various types are quite similar in properties (7), and all operate by electrophilic Abbreviations: CDP, copalyl diphosphate; GDP, geranyl diphosphate; reaction mechanisms, as do the prenyltransferases (8, 9). Com- GGDP, geranylgeranyl diphosphate; FDP, farnesyl diphosphate; LDP, prehensive treatment of the topic, especially enzymological and linalyl diphosphate. mechanistic aspects, has been provided recently (2–4), and the †Present address: Max-Planck-Institut fu¨r Chemische O¨ kologie, Tat- zendpromenade 1a, D-07745 Jena, Germany. e-mail: bohlmann@ice. mpg.de. © 1998 by The National Academy of Sciences 0027-8424y98y954126-8$2.00y0 §To whom reprint requests should be addressed. e-mail: croteau@ PNAS is available online at http:yywww.pnas.org. mail.wsu.edu. 4126 Downloaded by guest on September 27, 2021 Biochemistry: Bohlmann et al. Proc. Natl. Acad. Sci. USA 95 (1998) 4127 FIG. 3. Biosynthesis of representative sesquiterpenes from FDP. possible from a precursor with five additional carbon atoms [the monoterpenes number approximately 1,000, the sesquit- erpenes more than 7,000 (5)]. As with the monoterpenes, the formation of cyclohexanoid compounds, such as a-bisabolene, requires preliminary isomerization of the trans-farnesyl pre- cursor to the sesquiterpene analog of LDP, i.e., 3R-or3S- nerolidyl diphosphate, followed by ionization-dependent cy- clization (Fig. 3). The increased size of the farnesyl chain also FIG. 1. Organization of terpene biosynthesis in plants. DMADP, permits cyclization to 10- and 11-membered macrocycles such dimethylallyl diphosphate. IDP, isopentenyl diphosphate. as germacrene C (30) and g-humulene (31). Internal additions to the remaining double bonds of the initially formed cyclic A number of monoterpene synthases from angiosperms, carbocations also occur that, along with hydride shifts, methyl gymnosperms, and bryophytes have been partially purified and migrations, and Wagner–Meerwein rearrangements, permit characterized (7, 22, 23), and all have similar properties; native generation of a broad range of structures, including d-cadinene molecular mass in the 50- to 100-kDa range (either monomers (32), d-selinene (31), epi-aristolochene (33), vetispiradiene or homodimers), a requirement for a divalent metal ion 21 21 (31), and longifolene (31) (Fig. 3). Acyclic sesquiterpenes, such (usually Mg or Mn ), a pI value near 5.0, and a pH optimum as b-farnesene, are also derived from FDP by related synthases within a unit of neutrality. Monoterpene synthases are oper- (35, 36). The similarities in reaction mechanisms between the ationally soluble, although most are associated with plastids in plastidial monoterpene synthases and the cytosolic sesquiter- vivo (6, 20, 24). The synthases of gymnosperms are distinguish- pene synthases are paralleled by similarities in properties (7). able from their angiosperm counterparts by the requirement The diterpenoids are also widespread in the plant kingdom, for a monovalent cation (K1 preferred), a preference for Mn21 1 1 and they often are encountered in the resins of conifers, woody (or Fe2 ) over Mg2 as cofactor, and a generally higher pH legumes, composites, and members of the Euphorbiaceae. optimum. An interesting feature of several monoterpene syn- More than 3,000 different diterpenoid structures have been thases is their ability to produce multiple products (14, 25). For defined, all of which appear to be derived from GGDP (37). example, the (2)-pinene synthases from sage and grand fir The most common diterpene is phytol, the acyclic alcohol side produce both (2)-a- and (2)-b-pinene (20, 25–28). chain of chlorophyll. However, most diterpenoids are cyclic, All sesquiterpenes are derived from FDP (29), and the and there appear to be two major, and fundamentally differ- structural