Unearthing the roots of the terpenome

Abstract

Although terpenoid synthases catalyze the most complex reactions in biology, these enzymes appear to play little role in the chemistry of catalysis other than to trigger the ionization and chaperone the conformation of flexible isoprenoid substrates and carbocation intermediates through multistep reaction cascades. Fidelity and promiscuity in this chemistry (whether a terpenoid synthase generates one or several products), depends on the permissiveness of the active site template in chaperoning each step of an isoprenoid coupling or cyclization reaction. Structure-guided mutagenesis studies of terpenoid synthases such as farnesyl diphosphate synthase, 5-epi-aristolochene synthase, and gamma-humulene synthase suggest that the vast diversity of terpenoid natural products is rooted in the facile evolution of alpha-helical folds shared by terpenoid synthases in all forms of life.

Figure 1

Family tree of terpenomic diversity. Examples of monoterpenes (C₁₀), sesquiterpenes (C₁₅), diterpenes (C₂₀), and triterpenes (C₃₀) are shown. The reactions indicated are catalyzed by enzymes that share the fold of farnesyl diphosphate synthase. Isoprenoid groups are color-coded to indicate their biosynthetic fates, and newly formed carbon-carbon bonds are green. Abbreviations: BPPase, bornyl diphosphate synthase; DMAPP, dimethylallyl diphosphate; FPPase, farnesyl diphosphate synthase; GGPPase, geranylgeranyl diphosphate synthase; IPP, isopentenyl diphosphate; LSase, limonene synthase; PPO, diphosphate; SQSase, squalene synthase; TSase, trichodiene synthase; TxSase, taxadiene synthase. From Christianson DW: Science 316:60–61 (6 April 2007). Illustration: P. Huey. Reprinted with permission from AAAS.

Figure 2

Isoprenoid coupling reactions. (A) (top) Binding conformations of the unreactive DMAPP analogue dimethylallyl-S-thiolodiphosphate coordinated to three Mg²⁺ ions (gray) and isopentenyl diphosphate (IPP) in the active site of farnesyl diphosphate synthase from E. coli (beige) [12] superimposed on the binding conformation of geranylgeranyl diphosphate in the active site of geranylgeranyl diphosphate synthase from S. alba (blue) [13]. (bottom) Stereochemistry of the isoprenoid chain elongation reaction inferred from the superposition above (for clarity, enzyme residues are not shown). The observed binding conformations of DMAPP and IPP suggest that the pyrophosphate product mediates the stereospecific elimination of the C2-H_R proton of the isopentenyl moiety [12]. Reprinted with permission from reference 13. Copyright 2006 American Chemical Society. (B) Isoprenoid coupling reactions catalyzed by terpenoid synthases. Farnesyl diphosphate synthase (FPPase) catalyzes chain elongation reactions leading to the formation of geranyl diphosphate (GPP) and longer isoprenoid diphosphates. Chrysanthemyl diphosphate synthase catalyzes a cyclopropanation reaction leading to chrysanthemyl diphosphate (CPP). Chimeric synthases constructed from FPPase and CPPase generate the branching product lavandulyl diphosphate (LPP) as well as cyclobutanation products maconellyl diphosphate (MPP) and planococcyl diphosphate (PPP). From Thulasiram HV, Erickson HK, Poulter, CD: Science 316:73–76 (6 April 2007). Reprinted with permission from AAAS. (C) Products generated by CPPase-FPPase chimeras when incubated with DMAPP and IPP as indicated: farnesyl diphosphate, blue; geranyl diphosphate, gold; lavadulyl diphosphate, green; chrysanthemyl diphosphate, red; maconellyl diphosphate, pink; planococcyl diphosphate, orange. Synthase abbreviations are as follows: FPPase-M is G69E/E210Q FPPase and CPPase-M is M98I/E177D/D243A CPPase, constructs prepared to facilitate the preparation of chimeric enzymes; these modified synthases exhibit kinetic parameters comparable to those of the wild-type synthases. Chimeric synthases are indicated by their splice junctions, e.g., the c69f synthase contains the first 69 residues of CPPase-M and the remaining residues of FPPase-M (see also Figure 3A). From Thulasiram HV, Erickson HK, Poulter, CD: Science 316:73–76 (6 April 2007). Reprinted with permission from AAAS.

Figure 3

Terpenoid synthase folds. (A) The class I terpenoid synthase fold is illustrated by the α-bundle fold of FPPase from E. coli [12], colored violet-red to represent the successively larger segments of CPPase substituted for those of FPPase in the CPPase-FPPase chimeras outlined in Figure 2C [20]. For example, the c69f chimera contains the first 69 residues of CPPase-M (violet segment) and the remaining residues of FPPase-M (blue-red segments), and so forth. The FPPase fold is also shared by the catalytic domains of terpenoid synthases that generate larger and more complex products, such as bornyl diphosphate synthase (BPPase), trichodiene synthase (TSase), and squalene synthase (SQSase); these synthases are color-coded in identical fashion with FPPase to highlight their topological similarities. Reprinted with permission from reference 1. Copyright 2007 American Association for the Advancement of Science. (B) The class II terpenoid synthase fold is a double α-barrel conserved in nisin cyclase, farnesyl transferase, and both domains of lanosterol synthase. This fold is topologically distinct from the α-bundle fold of the class I terpenoid synthase. Reprinted from reference 33.

Figure 4

New terpenoid cyclase structures. (A) The structure of limonene synthase reveals a 2-domain architecture characteristic of plant terpenoid cyclases. 2-Fluorolinalyl diphosphate (red) and three Mn²⁺ ions (purple) bind to the C-terminal catalytic domain, which exhibits the α-bundle fold of a class I terpenoid synthase (green). The N-terminal domain (orange) has no known catalytic function yet exhibits a fold similar to the double α-barrel fold of a class II terpenoid cyclase. Reprinted with permission from reference 27. Copyright 2007 National Academy of Sciences, U.S.A. (B) Stereoview of the superposition of A. terreus aristolochene synthase structures in the open, unliganded conformation (blue) and the closed, Mg^2+₃-PP_i liganded conformation (green; Mg²⁺ ions are green spheres and the PP_i anion is red). Significant conformational changes are triggered in the indicated helices and loops, the most significant of which is the ligand-induced ordering of the H-α1 loop that caps the active site cleft. Reprinted in part with permission from reference 28. Copyright 2007 American Chemical Society. (C) Aristolochene synthase mechanism. Reprinted with permission from reference 28. Copyright 2007 American Chemical Society. (D) Stereoview of the enclosed active site surface contour of aristolochene synthase in the Mg^2+₃-PP_i complex; protein atoms are omitted for clarity. Aristolochene is modeled within the meshwork surface to illustrate that the three-dimensional contour of the active site is very product-like. Interestingly, the proximity of the PP_i O3 atom to aristolochene C6 and C8 atoms implicates the PP_i anion as a possible general acid/base in the cyclization mechanism. Reprinted with permission from reference 28. Copyright 2007 American Chemical Society.

Figure 5

Re-evolution of terpenoid cyclase specificity. (A) Cyclization reactions catalyzed by the promiscuous terpenoid cyclase, γ-humulene synthase; interconversion of the trans- and cis-farnesyl cation occurs through intermediate nerolidyl diphosphate (OPP = diphosphate). (B) Saturation mutagenesis of plasticity residues comprising the active site contour of γ-humulene synthase reveals that only 2–5 amino acid substitutions are required to redirect biosynthesis to form an alternative predominant product as detected using gas chromatography. Product numbers are indicated in (A). Reprinted with permission from reference 46. Copyright 2006 Nature Publishing Group (http://www.nature.com/nature/).