4-Aldrithiol-based photometric assay for detection of methylthioalkylmalate synthase activity.

In Brassica plants, glucosinolates are a diverse class of natural products, of which aliphatic methionine-derived glucosinolates are the most abundant form. Their structural diversity comes from the elongation of some side-chains by up to 9 carbons, which, after the formation of the core glucosinolate structure, can undergo further chemical modifications. Methylthioalkylmalate synthase (MAMS) catalyzes the iterative elongation process for aliphatic methionine-derived glucosinolates. Most biochemical studies on MAMS have been performed using liquid chromatography/mass spectrometry (LC/MS)-based assays or high-performance liquid chromatography (HPLC)-based assays. The LC/MS- and HPLC-based methods are endpoint assays, which cannot be monitored in real time and require a laborious process for data collection. These analytical methods are inefficient for performing multiple enzymatic assays needed to determine steady-state kinetic parameters or for mechanistic evaluation of pH-dependence and kinetic isotope effect studies. Although the function of MAMS has long been defined, there is a gap in knowledge as it pertains to biochemical characterization of this plant enzyme. Part of this may be due to the lack of efficient methods that can be used for this type of research. This chapter describes a continuous photometric assay to track MAMS activity in real time using the 4-aldrithiol reagent for reaction detection.

A Functional Switch Between Asperfumene and Fusicoccadiene Synthase and Entrance to Asperfumene Biosynthesis through a Vicinal Deprotonation-Reprotonation Process.

The diterpene synthase AfAS was identified from Aspergillus fumigatiaffinis. Its amino acid sequence and-according to a structural model-active site architecture are highly similar to those of the fusicocca-2,10(14)-diene synthase PaFS, but AfAS produces a structurally much more complex diterpene with a novel 6-5-5-5 tetracyclic skeleton called asperfumene. The cyclisation mechanism of AfAS was elucidated through isotopic labelling experiments and DFT calculations. The reaction cascade proceeds in its initial steps through similar intermediates as for the PaFS cascade, but then diverges through an unusual vicinal deprotonation-reprotonation process that triggers a skeletal rearrangement at the entrance to the steps leading to the unique asperfumene skeleton. The structural model revealed only one major difference between the active sites: The PaFS residue F65 is substituted by I65 in AfAS. Intriguingly, site-directed mutagenesis experiments with both diterpene synthases revealed that position 65 serves as a bidirectional functional switch for the biosynthesis of tetracyclic asperfumene versus structurally less complex diterpenes.

Discovery of a terpene synthase synthesizing a nearly non-flexible eunicellane reveals the basis of flexibility.

Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies.

Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids.

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Comprehensive analysis of CXXX sequence space reveals that Saccharomyces cerevisiae GGTase-I mainly relies on a2X substrate determinants.

Many proteins undergo a post-translational lipid attachment, which increases their hydrophobicity, thus strengthening their membrane association properties or aiding in protein interactions. Geranylgeranyltransferase-I (GGTase-I) is an enzyme involved in a 3-step post-translational modification (PTM) pathway that attaches a 20-carbon lipid group called geranylgeranyl at the carboxy-terminal cysteine of proteins ending in a canonical CaaL motif (C-cysteine, a-aliphatic, L-often leucine, but can be phenylalanine, isoleucine, methionine, or valine). Genetic approaches involving 2 distinct reporters were employed in this study to assess Saccharomyces cerevisiae GGTase-I specificity, for which limited data exist, toward all 8,000 CXXX combinations. Orthogonal biochemical analyses and structure-based alignments were also performed to better understand the features required for optimal target interaction. These approaches indicate that yeast GGTase-I best modifies the Cxa[L/F/I/M/V] sequence that resembles but is not an exact match for the canonical CaaL motif. We also observed that minor modification of noncanonical sequences is possible. A consistent feature associated with well-modified sequences was the presence of a nonpolar a2 residue and a hydrophobic terminal residue, which are features recognized by mammalian GGTase-I. These results thus support that mammalian and yeast GGTase-I exhibit considerable shared specificity.

Gibberellin-biosynthetic ent-kaurene synthases in higher plants do not require their non-catalytic domains for the catalysis.

ent-Kaurene is a biosynthetic intermediate diterpene of phytohormone gibberellins, and is biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate (ent-CDP). The successive cyclization is catalyzed by two distinct diterpene synthases, ent-CDP synthase (ent-CPS) and ent-kaurene synthase (KS). Homologs of these diterpene synthase genes have been reported to be involved in the biosynthesis of specialized-metabolic diterpenoids for defense in several plant species, including rice (Oryza sativa). These diterpene synthases consist of three domains, αßγ domains. Active sites of ent-CPS exist at the interface of ß and γ domain, while those of KS are located within the α domain. We herein carried out domain-deletion experiments using several KSs and KS like enzymes (KSLs) to obtain insights into the roles of domains other than active-site domains. As previously reported in taxadiene synthase, deletion of γ or ßγ domains drastically decreased activities of specialized-metabolic OsKSL5, OsKSL8, OsKSL7 and OsKSL10 in O. sativa. However, unexpectedly, only α domains of several gibberellin-biosynthetic KSs, including OsKS1 in O. sativa, AtKS in Arabidopsis thaliana, TaKS in wheat (Triticum aestivum) and BdKS1 in Brachypodium distachyon, retained their original functions. Additionally, the specialized-metabolic OsKSL4, which is closely related to OsKS1, also functioned without its ßγ domains. Domain-swapping experiments showed that replacing ßγ domains in OsKSL7 with those from other KS/KSLs retained the OsKSL7 activity. Moreover, deletion of ßγ domains of bifunctional PpCPS/KS in moss (Physcomitrella patens) drastically impaired its KS-related activity. Thus, we demonstrate that monofunctional gibberellin-biosynthetic KSs are the unique diterpene synthases that retain their functions without ßγ domains.

Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanum.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

Mechanistic docking in terpene synthases using EnzyDock.

Terpene Synthases (TPS) catalyze the formation of multicyclic, complex terpenes and terpenoids from linear substrates. Molecular docking is an important research tool that can further our understanding of TPS multistep mechanisms and guide enzyme design. Standard docking programs are not well suited to tackle the unique challenges of TPS, like the many chemical steps which form multiple stereo-centers, the weak dispersion interactions between the isoprenoid chain and the hydrophobic region of the active site, description of carbocation intermediates, and finding mechanistically meaningful sets of docked poses. To address these and other unique challenges, we developed the multistate, multiscale docking program EnzyDock and used it to study many TPS and other enzymes. In this review we discuss the unique challenges of TPS, the special features of EnzyDock developed to address these challenges and demonstrate its successful use in ongoing research on the bacterial TPS CotB2.

Understanding mechanisms of terpene synthases using substrate analogs.

Terpene synthases (TS) transform achiral prenyl substrates into elaborate hydrocarbon scaffolds with multiple stereocenters through a series of cyclization reactions and carbon skeleton rearrangements. The reactions involve high-energy carbocation intermediates that must be stabilized by the enzyme along the pathway to the desired products. A variety of substrate analogs have been used to investigate TS mechanism. This article will focus on a class of analogs which strategically replace hydrogen atoms with fluorine to inhibit the generation of specific carbocation intermediates. We will explore the synthesis and use of the analogs to study TS mechanism.

Production of non-natural terpenoids through chemoenzymatic synthesis using substrate analogs.

Chemoenzymatic synthesis of non-natural terpenes using the promiscuous activity of terpene synthases allows for the expansion of the chemical space of terpenoids with potentially new bioactivities. In this report, we describe protocols for the preparation of a novel aphid attractant, (S)-14,15-dimethylgermacrene D, by exploiting the promiscuity of (S)-germacrene D synthase from Solidago canadensis and using an engineered biocatalytic route to convert prenols to terpenoids. The method uses a combination of five enzymes to carry out the preparation of terpenoid semiochemicals in two steps: (1) diphosphorylation of five or six carbon precursors (prenol, isoprenol and methyl-isoprenol) catalyzed by Plasmodium falciparum choline kinase and Methanocaldococcus jannaschii isopentenyl phosphate kinase to form DMADP, IDP and methyl-IDP, and (2) chain elongation and cyclization catalyzed by Geobacillus stearothermophilus (2E,6E)-farnesyl diphosphate synthase and S. canadensis (S)-germacrene D synthase to produce (S)-germacrene D and (S)-14,15-dimethylgermacrene D. Using this method, new non-natural terpenoids are readily accessible and the approach can be adopted to produce different terpene analogs and terpenoid derivatives with potential novel applications.

Docking carbocations into terpene synthase active sites using chemically meaningful constraints-The TerDockin approach.

Terpenes are a diverse class of natural products which have long been sought after for their chemical properties as medicine, perfumes, and for food flavoring. Computational docking studies of terpene mechanisms have been a challenge due to the lack of strong directing groups which many docking programs rely on. In this chapter, we dive into our computational method Terdockin (Terpene-Docking) as a successful methodology in modeling terpene synthase mechanisms. This method could also be used as inspiration for any multi-ligand docking project.

Isotopic labelings for mechanistic studies.

The intricate mechanisms in the biosynthesis of terpenes belong to the most challenging problems in natural product chemistry. Methods to address these problems include the structure-based site-directed mutagenesis of terpene synthases, computational approaches, and isotopic labeling experiments. The latter approach has a long tradition in biosynthesis studies and has recently experienced a revival, after genome sequencing enabled rapid access to biosynthetic genes and enzymes. Today, this allows for a combined approach in which isotopically labeled substrates can be incubated with recombinant terpene synthases. These clearly defined reaction setups can give detailed mechanistic insights into the reactions catalyzed by terpene synthases, and recent developments have substantially deepened our understanding of terpene biosynthesis. This chapter will discuss the state of the art and introduce some of the most important methods that make use of isotopic labelings in mechanistic studies on terpene synthases.

Ancestral terpene cyclases: From fundamental science to applications in biosynthesis.

Terpenes constitute one of the largest family of natural products with potent applications as renewable platform chemicals and medicines. The low activity, selectivity and stability displayed by terpene biosynthetic machineries can constitute an obstacle towards achieving expedient biosynthesis of terpenoids in processes that adhere to the 12 principles of green chemistry. Accordingly, engineering of terpene synthase enzymes is a prerequisite for industrial biotechnology applications, but obstructed by their complex catalysis that depend on reactive carbocationic intermediates that are prone to undergo bifurcation mechanisms. Rational redesign of terpene synthases can be tedious and requires high-resolution structural information, which is not always available. Furthermore, it has proven difficult to link sequence space of terpene synthase enzymes to specific product profiles. Herein, the author shows how ancestral sequence reconstruction (ASR) can favorably be used as a protein engineering tool in the redesign of terpene synthases without the need of a structure, and without excessive screening. A detailed workflow of ASR is presented along with associated limitations, with a focus on applying this methodology on terpene synthases. From selected examples of both class I and II enzymes, the author advocates that ancestral terpene cyclases constitute valuable assets to shed light on terpene-synthase catalysis and in enabling accelerated biosynthesis.

Identification and functional/structural analyses of large terpene synthases.

Large terpene synthases (large-TSs) are a new family of TSs. The first large-TS discovered was from Bacillus subtilis (BsuTS), which is involved in the biosynthesis of a C35 sesquarterpene. Large-TSs are the only enzymes that enable the biosynthesis of sesquarterpenes and do not share any sequence homology with canonical Class I and II TSs. Thus, the investigation of large-TSs is promising for expanding the chemical space in the terpene field. In this chapter, we describe the experimental methods used for identifying large-TSs, as well as their functional and structural analyses. Additionally, several enzymes related to the biosynthesis of large-TS substrates have been described.

Structural biology of terpene synthases.

Structural biology research of terpene synthases (TSs) has provided a useful basis to understand their catalytic mechanisms in producing diverse terpene products with polycyclic ring systems and multiple chiral centers. However, compared to the large numbers of>95,000 terpenoids discovered to date, few structures of TSs have been solved and the understanding of their catalytic mechanisms is lagging. We here (i) introduce the basic catalytic logic, the structural architectures, and the metal-binding conserved motifs of TSs; (ii) provide detailed experimental procedures, in gene cloning and plasmid construction, protein purification, crystallization, X-ray diffraction data collection and structural elucidation, for structural biology research of TSs; and (iii) discuss the prospects of structure-based engineering and de novo design of TSs in generating valuable terpene molecules, which cannot be easily achieved by chemical synthesis.

Vanadium haloperoxidases as noncanonical terpene synthases.

Vanadium-dependent haloperoxidases (VHPOs) are a unique family of enzymes that utilize vanadate, an aqueous halide ion, and hydrogen peroxide to produce an electrophilic halogen species that can be incorporated into electron rich organic substrates. This halogen species can react with terpene substrates and trigger halonium-induced cyclization in a manner reminiscent of class II terpene synthases. While not all VHPOs act in this capacity, several notable examples from algal and actinobacterial species have been characterized to catalyze regio- and enantioselective reactions on terpene and meroterpenoid substrates, resulting in complex halogenated cyclic terpenes through the action of single enzyme. In this article, we describe the expression, purification, and chemical assays of NapH4, a difficult to express characterized VHPO that catalyzes the chloronium-induced cyclization of its meroterpenoid substrate.

Function Switch of a Fungal Sesterterpene Synthase through Molecular Dynamics Simulation Assisted Alteration of an Aromatic Residue Cluster in the Active Pocket of PfNS.

Terpene synthases (TPSs) play pivotal roles in generating diverse terpenoids through complex cyclization pathways. Protein engineering of TPSs offers a crucial approach to expanding terpene diversity. However, significant potential remains untapped due to limited understanding of the structure-function relationships of TPSs. In this investigation, using a joint approach of molecular dynamics simulations-assisted engineering and site-directed mutagenesis, we manipulated the aromatic residue cluster (ARC) of a bifunctional terpene synthase (BFTPS), Pestalotiopsis fici nigtetraene synthase (PfNS). This led to the discovery of previously unreported catalytic functions yielding different cyclization patterns of sesterterpenes. Specifically, a quadruple variant (F89A/Y113F/W193L/T194W) completely altered PfNS's function, converting it from producing the bicyclic sesterterpene nigtetraene to the tricyclic ophiobolin F. Additionally, analysis of catalytic profiles by double, triple, and quadruple variants demonstrated that the ARC functions as a switch, unprecedently redirecting the production of 5/11 bicyclic (Type B) sesterterpenes to 5/15 bicyclic (Type A) ones. Molecular dynamics simulations and theozyme calculations further elucidated that, in addition to cation-π interactions, C-H⋅⋅⋅π interactions also play a key role in the cyclization patterns. This study offers a feasible strategy in protein engineering of TPSs for various industrial applications.

[Functional characterization of ent-kaurane-type diterpenoid synthases from Stellera chamaejasme].

Sequential catalysis by ent-copalyl diphosphate(CPS) and ent-kaurene synthase(KS) is a critical step for plants to initiate the biosynthesis of gibberellin with geranylgeranyl pyrophosphate(GGPP) as the substrate. This study mined the transcriptome data of Stellera chamaejasme and cloned two key diterpene synthase genes, SchCPS and SchKS, involved in the gibberellin pathway. The two genes had the complete open reading frames of 2 595 bp and 1 701 bp, encoding two hydrophilic proteins composed of 864 and 566 amino acid residues and with the relative molecular mass of 97.9 kDa and 64.6 kDa and the theoretical isoelectric points of 5.61 and 6.12, respectively. Sequence comparison and phylogenetic tree showed that SchCPS contained LHS, PNV, and DxDD motifs conserved in the CPS family and was categorized in the TPS-c subfamily, while SchKS contained DDxxD, NSE/DTE and PIx motifs conserved in the KS family and was categorized in the TPS-e subfamily. Functional validation showed that SchCPS catalyzed the protonation and cyclization of GGPP to ent-CPP, while SchKS acted on ent-CPP dephosphorylation and re-cyclization to ent-kaurene. In this study, the full-length sequences of SchCPS and SchKS were cloned and functionally verified for the first time, which not only enriched the existing CPS and KS gene libraries but also laid a foundation for the cloning and biosynthesis pathway analysis of more genes involved in the synthesis of active components in S. chamaejasme.

Essential residues in diterpene synthases for biosynthesis of oryzalexins A-F in rice phytoalexin.

Cultivated rice (Oryza sativa) produces a variety of diterpenoid-type phytoalexins. Diterpene synthase genes that are responsible for the biosynthesis of momilactones, phytocassanes, and oryzalexins have been identified in O. sativa cv. Nipponbare. OsKSL10 (Os12t0491800 in RAP and LOC_Os12g30824 in MSU) was previously identified as an enzyme catalyzing the conversion of ent-copalyl diphosphate to ent-sandaracopimaradiene for the production of oryzalexins A to F. Our previous study on Oryza rufipogon, a wild progenitor of Asian cultivated rice, showed that both OrKSL10 and OrKSL10ind from O. rufipogon accessions W1943 and W0106, respectively, closely related to the japonica and indica subspecies, converted ent-copalyl diphosphate to ent-miltiradiene. Thus, the functional conversion of ent-miltiradiene synthase into ent-sandaracopimaradiene synthase is implied to have occurred through natural amino acid mutations, the details of which have not been elucidated. In this study, we show that introduction of A654G substitution into OrKSL10 significantly alters its function into more closely resembling that of OsKSL10. Moreover, double substitution V546I/A654G almost completely converts the function of OrKSL10 into that of OsKSL10. On the other hand, the reversed substitution I546V/G654A was insufficient to convert the function of OsKSL10 into OrKSL10, indicating the introduction of additional substitution S522I is required for the functionality of OsKSL10. Lastly, point mutations at the 654A residue in OrKSL10 suggest that hydrophobic side chains at this position have a negative influence on the production of ent-sandaracopimaradiene.