The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [Chen, M., Chou, W. K. W., Toyomasu, T., Cane, D. E., Christianson, D. W. (2016) ACS Chem. Biol. 11, 889–899]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20 diterpene products and three C25 sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with 3 Mg2+ ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed based on ~36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

Crystal structures of terpenoid cyclases reveal assemblies of three basic domains designated α, β, and γ. While the biosynthesis of cyclic monoterpenes (C10) and sesquiterpenes (C15) most often involves enzymes with α or αβ domain architecture, the biosynthesis of cyclic diterpenes (C20), sesterterpenes (C25), and triterpenes (C30) can involve enzymes with α, αα, βγ, or αβγ domain architecture. Indeed, some enzymes of terpenoid biosynthesis are bifunctional, with distinct active sites that catalyze sequential reactions. Interestingly, some of these enzymes oligomerize to form dimers, tetramers, and hexamers. Not only can such assemblies enable enzyme regulation by allostery, but they can also provide a modest enhancement of terpenoid product flux through proximity channeling or cluster channeling. The mixing and matching of functional terpenoid cyclase domains through tertiary and/or quaternary structure may also comprise an evolutionary strategy for facile product diversification.

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The class I terpenoid cyclase epi‐isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi‐isozizaene as the predominant sesquiterpene cyclization product, along with at least 5 minor sesquiterpene products. The active site contour of EIZS serves as a template that enforces a particular substrate conformation and chaperones carbocation intermediates through a well‐defined mechanistic sequence. We have engineered the active site of EIZS by remolding its hydrophobic contour through site‐directed mutagenesis in order to decipher the code that relates the three‐dimensional contour of the active site with the products generated by the enzyme. Significantly, the remolded cyclization template in EIZS mutants reprograms the reaction cascade not only to adjust the proportions of products generated by the wild‐type enzyme, but also to generate completely new products of diverse structure. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100‐fold losses of catalytic activity, suggesting that product diversity, and not necessarily catalytic efficiency, is a critical driver in the evolution of terpenoid biosynthesis. Grant Funding Source: Supported by GM56838