Cyclization Enzymes in the Biosynthesis of Monoterpenes ... Cyclization Enzymes in the Biosynthesis

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  • Terpene synthases catalyze the first committed steps in the biosynthesis of monoterpenes, ses- quiterpenes, and diterpenes. An overview is presented of the enzymology and mechanism of these terpene synthases, and their molecular cloning, expression, and sequence analysis. Detailed structural and functional evaluation of four representative monoterpene, sesquiter- pene, and diterpene synthases is also presented.

    Keywords. Terpene, Monoterpene, Sesquiterpene, Diterpene, Cyclase, Geranyl diphosphate, Farnesyl diphosphate, Geranylgeranyl diphosphate

    1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

    2 Prenyl Transferases and Terpenoid Synthases . . . . . . . . . . . . . . 58

    3 Monoterpene Synthases . . . . . . . . . . . . . . . . . . . . . . . . . . 59

    3.1 Mechanism and Stereochemistry . . . . . . . . . . . . . . . . . . . . . 61 3.2 Limonene Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

    4 Sesquiterpene Synthases . . . . . . . . . . . . . . . . . . . . . . . . . 72

    4.1 G-Humulene Synthase and d-Selinene Synthase . . . . . . . . . . . . . 77

    5 Diterpene Synthases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

    5.1 Abietadiene Synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

    6 Tertiary Structure of 5-epi-Aristolochene Synthase and Prediction of Structure/Function Relationships . . . . . . . . . . . . . . . . . . . 89

    7 Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

    8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

    Cyclization Enzymes in the Biosynthesis of Monoterpenes, Sesquiterpenes, and Diterpenes

    Edward M. Davis · Rodney Croteau

    Institute of Biological Chemistry, Washington State University, Pullman, WA 99164–6340, USA E-mail: croteau@mail.wsu.edu

    Topics in Current Chemistry, Vol. 209 © Springer-Verlag Berlin Heidelberg 2000

  • Abbreviations

    CPP copalyl diphosphate DMAPP dimethylallyl diphosphate FPP farnesyl diphosphate GPP geranyl diphosphate GGPP geranylgeranyl diphosphate Hm-VS Hyoscyamus muticus vetispiradiene synthase IPP isopentenyl diphosphate KS kaurene synthase LPP linalyl diphosphate NPP nerolidyl diphosphate Nt-EAS Nicotiana tabacum 5-epi-aristolochene synthase tps terpene synthase WM Wagner-Meerwein rearrangement

    1 Introduction

    The terpenes are a structurally diverse and widely distributed family of natural products containing well over 30,000 defined compounds identified from all kingdoms of life [1]. The majority of terpenes have been isolated from plants where they serve a broad range of roles in primary metabolism (including se- veral plant hormones and the most abundant plant terpenoid, phytol, the side chain of the photosynthetic pigment chlorophyll) and in ecological interactions (as chemical defenses against herbivores and pathogens, pollinator attractants, allelopathic agents, etc.) (Table 1). Many terpenes are of economic importance, including the essential oils, carotenoid pigments and natural rubber. Terpenes are constructed by the repetitive joining of branched C5 isoprenoid units and are categorized according to size with the C10 family, historically considered the smallest, named the monoterpenes. The nomenclature thus evolved with the sesquiterpenes (C15), diterpenes (C20), sesterterpenes (C25), triterpenes (C30), tetraterpenes (C40), and polyterpenes (>C40). Isoprene, discovered as a natural product in the 1960’s, was thus considered a hemiterpene (C5). The meroterpenes consist of a terpene-derived unit attached to a non-terpene moiety; examples include chlorophyll, plastoquinone, certain indole alkaloids, and prenylated proteins.

    All terpenoids are derived from the universal precursor isopentenyl di- phosphate (IPP) which, following isomerization to dimethylallyl diphosphate (DMAPP) by IPP isomerase, is sequentially elongated by prenyltransferases to geranyl diphosphate (GPP, C10), farnesyl diphosphate (FPP, C15), and geranyl- geranyl diphosphate (GGPP, C20) (Scheme 1). These acyclic intermediates function at the branch points of isoprenoid metabolism from which the mono- terpenes (from GPP), sesquiterpenes (from FPP), and diterpenes (from GGPP) diverge through the action of the terpenoid synthases. The terpenoid synthases, often called cyclases since the products of the reaction are most often cyclic,

    54 E. M. Davis · R. Croteau

  • Cyclization Enzymes in the Biosynthesis of Monoterpenes, Sesquiterpenes, and Diterpenes 55

    Table 1. Classification, structures, and physiological and ecological roles of selected terpenoids

    Monoterpenes medicinal limonene

    camphor

    flavoring menthol

    Sesquiterpenes perfumary raw material patchoulol

    phytohormone abscisic acid

    antibiotic pentalenolactone

    Diterpenes anti-cancer drug taxol

    phytoalexin casbene

    phytohormone gibberellin

    Triterpene membrane component b-sitosterol

    Tetraterpene plant pigment b-carotene (provitamin A)

    Meroterpenes photosynthesis chlorophyll

    electron transport plastoquinone

    Polyterpene commercial rubber (industrial raw material)

  • serve to convert the three acyclic branch point intermediates to the parent skeletons of the various monoterpene, sesquiterpene, and diterpene types. This family of enzymes, which forms the focus of this review, not only catalyzes the committed step in the biosynthesis of most terpenoids but is also largely responsible for the great structural diversity encountered in this class of natural products.

    The past five years have witnessed a major reevaluation of the biogenetic origin of IPP as the universal precursor of terpenoids. Since its elucidation in the late 1950’s [2], the classical acetate-mevalonate pathway (Scheme 2A) has been considered the only biosynthetic route to terpenoids. However, a mevalonate- independent pathway, with its origins in the condensation of pyruvate (via hydroxyethyl thiamine pyrophosphate) and glyceraldehyde-3-phosphate to form 1-deoxyxylose-5-phosphate (Scheme 2B), has been discovered to operate in certain bacteria [3] and, more recently, in the plastids of both lower and higher plants [4–6]. It is now clear that terpenoid biosynthesis in plants is compartmentalized, with the sesquiterpenes (and triterpenes) arising

    56 E. M. Davis · R. Croteau

    Scheme 1

  • Cyclization Enzymes in the Biosynthesis of Monoterpenes, Sesquiterpenes, and Diterpenes 57

    A

    Scheme 2

  • in the cytosol [7] via the acetate-mevalonate pathway, and the monoterpenes and diterpenes (and tetraterpenes) arising in the plastids [6–9] via the still incompletely understood mevalonate-independent pathway of bacterial endosymbiotic origin. Each compartment contains IPP isomerase [10] and the requisite prenyltransferases for construction of the appropriate pre- cursors.

    2 Prenyltransferases and Terpenoid Synthases

    The prenyltransferases catalyze the head-to-tail condensation of IPP with either DMAPP (geranyl diphosphate synthase), GPP (farnesyl diphosphate synthase), or FPP (geranylgeranyl diphosphate synthase), although FPP syn- thase and GGPP synthase can also utilize only IPP and DMAPP as the initial substrates in multistep elongation sequences via bound intermediates [11]. The electrophilic reaction mechanisms of the prenyltransferases involves initial ionization of the allylic diphosphate ester substrate to form a charge- delocalized carbocation which undergoes C4¢-C1 coupling via the terminal double bond of IPP to generate a tertiary carbocation followed by deprotona- tion to complete the reaction (Scheme 1). The terpenoid synthase reaction may be regarded as the intramolecular analogue of the intermolecular electro- philic coupling reaction catalyzed by the prenyltransferases, although the detailed reaction variants encountered in the former case are far greater than in the latter [11, 12]. More than a common mechanistic link was established between the prenyltransferases and the terpenoid synthases (cyclases) with the discovery that avian FPP synthase is capable of generating cyclic C15 olefins as products with FPP as substrate [12, 13]. The two enzyme types also exhibit notable conservation in primary sequence and physical characteristics (see below).

    As might be expected based on the similarities in reaction mechanism, the prenyltransferases and terpenoid synthases share a number of common prop- erties. Both are functionally soluble, acidic enzymes, with native sizes in the 35–80 kDa range, require only a divalent metal ion for catalysis, and exhibit Michaelis constants for the prenyl substrate that rarely exceed 10 µM and turn- over numbers typically ranging from 0.03 to 0.3 s–1 [14]. The similarities between the prenyltransferases and the terpenoid synthases are clearly of evolutionary significance; however, it is equally clear that the diversity in type and mechan- istic complexity set the terpenoid synthases apart from the more conservative prenyltransferases. The typical cyclization reaction involves ionization of the prenyl diphosphate substrate with intramolecular attack by a double bond of the prenyl chain at C1 to yield a cyclic carbocationic intermediate. Subsequent steps may involve further internal additions via the remaining double bonds to gene- rate additional rings, and may be accompanied by hydride shifts, methyl migra- tions and Wagner-Meerwein rearrangements (WM) before termination of the reaction by deprotonation or nucleophile (often water) capture. The terpenoid synthases commonly produce multiple products, although high fidelity