Terpenoid synthase structures: a so far incomplete view of complex catalysis

The complexity of terpenoid natural products has drawn significant interest, particularly since their common (poly)isoprenyl origins were discovered. Notably, much of this complexity is derived from the highly variable cyclized and/or rearranged nature of the observed hydrocarbon skeletal structures. Indeed, at least in some cases it is difficult to immediately recognize their derivation from poly-isoprenyl precursors. Nevertheless, these diverse structures are formed by sequential elongation to acyclic precursors, most often with subsequent cyclization and/or rearrangement. Strikingly, the reactions used to assemble and diversify terpenoid backbones share a common carbocationic driven mechanism, although the means by which the initial carbocation is generated does vary. High-resolution crystal structures have been obtained for at least representative examples from each of the various types of enzymes involved in producing terpenoid hydrocarbon backbones. However, while this has certainly led to some insights into the enzymatic structure–function relationships underlying the elongation and simpler cyclization reactions, our understanding of the more complex cyclization and/or rearrangement reactions remains limited. Accordingly, selected examples are discussed here to demonstrate our current understanding, its limits, and potential ways forward

Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants

Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Here we report mutational investigation of this discrepancy in a model plant diterpene cyclase, abietadiene synthase from Abies grandis (AgAS). Substitution of the corresponding Thr in AgAS with Ser or Gly decreased catalytic activity much less than substitution with Ala. We speculate that the ability of Gly to partially restore activity relative to Ala substitution for Ser/Thr stems from the associated reduction in steric volume enabling a water molecule to substitute for the hydroxyl group from Ser/Thr, potentially in a divalent metal ion coordination sphere. In any case, our results are consistent with the observed conservation pattern for this putative second divalent metal ion binding motif in plant TPS

ENGINEERING NOVEL TERPENE PRODUCTION PLATFORMS IN THE YEAST SACCHAROMYCES CEREVISIAE

The chemical diversity and biological activities of terpene and terpenoids have served in the development of new flavors, fragrances, medicines and pesticides. While terpenes are made predominantly by plants and microbes in small amounts and as components of complex mixtures, chemical synthesis of terpenes remains technically challenging, costly and inefficient. In this dissertation, methods to create new yeast lines possessing a dispensable mevalonate biosynthetic pathway wherein carbon flux can be diverted to build any chemical class of terpene product are described. The ability of this line to generate diterpenes was next investigated. Using a 5.5 L fed bath fermentation system, about 569 mg/L kaurene and approximately 207 mg/L abietadiene plus 136 mg/L additional isomers were achieved. To engineer more highly modified diterpenes might have greater industrial, agricultural or medicinal applications, kaurenoic acid production reached 514 mg/L with byproduct kaurene and kaurenal at 71.7mg/L and 20.1mg/L, respectively, in fed batch fermentation conditions. Furthermore, ZXM lines for engineer monoterpene and ZXB lines for engineer triterpene were generated by additional specific genomic modification, 84.76 ±13.2 mg/L linalool, 20.54±3.8 mg/L nerolidol and 297.7mg/L squalene were accumulate in ZXM144 line ana ZXB line, respectively, in shake flask conditions

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product

Insights into Diterpene Cyclization from Structure of Bifunctional Abietadiene Synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg2+ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities

Investigating reactions catalyzed by terpene synthases in a novel model system

Terpene synthases are a set of enzymes that initiate the biosynthetic production of the largest class of known natural products. However, not much is known about how these enzymes carry out their complex cyclization reactions to form polycyclic olefins from linear hydrocarbon diphosphates such as geranylgeranyl diphosphate.;Since rice is known to produce many diterpenoid natural products and the rice genome was recently published, sequence information for many putative diterpene synthases was available. This provided the basis for functional characterization of the diterpene synthases from rice (Oryza sativa ssp. Indica). An enzyme producing syn-pimaradiene was identified, followed by those producing syn-stemodene, ent-kaurene, ent-isokaurene, ent-sandaracopimaradiene, ent-cassadiene, and syn-stemarene. At the same time another group published similar results, however one of the reported ent-isokaurene synthases was reported as ent-pimaradiene synthase by another research group.;Noting this difference in product with only the three amino acid differences in the active site, experiments were carried out leading to discovery of a single amino acid residue that switches product profile completely to pimaradienes from kaurenes, OsKSL5i:I664T. This switch was found not only in the originally targeted isokaurene synthase, OsKSL5, but also in the second reported isokaurene synthase from rice and kaurene synthases from rice and Arabidopsis. Further expounding on this idea, a similar amino acid difference was noted in diterpene synthases of conifers. This amino acid difference, A723S in abietadiene synthase, was a switch, too, with the product profile switching from \u3e95% abietanes to \u3e95% pimaranes. Utilizing a combination of functional genomics, metabolic engineering, macromolecular modeling, and enzyme biochemistry a basis for further investigations into how terpene synthases work has been provided

To Gibberellins and Beyond! Surveying the Evolution of (Di)Terpenoid Metabolism

The diterpenoids are classically defined by their composition, four isoprenyl units (20 carbons), and are generally derived from [E,E,E]-geranylgeranyl diphosphate (GGPP). Such metabolism seems to be ancient and has been extensively diversified, with ~12,000 diterpenoid natural products known. Particularly notable are the gibberellin phytohormones, whose requisite biosynthesis has provided a genetic reservoir giving rise to not only a large super-family of ~7,000 diterpenoids, but to some degree all plant terpenoid natural products. This review focuses on the diterpenoids, particularly the defining biosynthetic characteristics of the major superfamilies defined by the cyclization and/or rearrangement of GGPP catalyzed by diterpene synthases/ cyclases, although some discussion also is provided of the important subsequent elaboration in those few cases where molecular genetic information is available. In addition, the array of biological activity providing the selective pressure driving the observed gene family expansion and diversification, along with biosynthetic gene clustering, will be discussed as well

On the biosynthesis of labdane-related diterpenoids by class I synthases and P450s: a

Due to their abundance in secondary metabolites, plants are a rich source of chemical diversity. Terpenoids comprise the largest class of natural products, yet many of the biosynthetic pathways leading to their production remain under explored. Using rice as a model system, the biosynthesis of labdane-related class I synthases and P450s was explored through the development of a facile, modular metabolic engineering system. Herein, details of the characterization of the rice class I labdane-related synthase family and CYP76M7 are included. Accordingly, it was found that rice possesses a stemodene synthase, bi-selective syn-labdatriene/ent-sandaracopimaradiene synthase, and a cassadiene C11 hydroxylating P450. Phytochemical analysis of rice extracts revealed the presence of the characterized diterpenes made by class I synthases. Additionally, it was found that the class I synthases exhibit a high degree of plasticity with respect to substrate selection and product outcome, including the ability of OsKSL4 T696I to produce aphidicolene. Furthermore, the first bacterial class I synthases involved in gibberellin production has been identified and characterized. Kaurene oxidase is the first cytochrome P450 involved in the biosynthesis of gibberellin growth hormones. Oxygen labeling experiments revealed this multifunctional P450 undergoes successive hydroxylation reactions with retention of intermediates through a gem-diol to form a carboxylic acid. The studies presented in this thesis detail the development of a metabolic engineering system to investigate terpenoid biosynthesis, the characterization of class I synthases and P450s, and mechanistic investigations of class I synthases and P450s involved in labdane-related biosynthesis