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Modulation of inherent dynamical tendencies of the bisabolyl cation via preorganization in epi-isozizaene synthase.

ABSTRACT: The relative importance of preorganization, selective transition state stabilization and inherent reactivity are assessed through quantum chemical and docking calculations for a sesquiterpene synthase (epi-isozizaene synthase, EIZS). Inherent reactivity of the bisabolyl cation, both static and dynamic, appears to determine the pathway to product, although preorganization and selective binding of the final transition state structure in the multi-step carbocation cascade that forms epi-isozizaene appear to play important roles.

SUBMITTER: Pemberton RP

PROVIDER: S-EPMC5645776 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Modulation of inherent dynamical tendencies of the bisabolyl cation via preorganization in epi-isozizaene synthase.

Pemberton Ryan P RP Ho Krystina C KC Tantillo Dean J DJ

Chemical science 20150202 4

The relative importance of preorganization, selective transition state stabilization and inherent reactivity are assessed through quantum chemical and docking calculations for a sesquiterpene synthase (epi-isozizaene synthase, EIZS). Inherent reactivity of the bisabolyl cation, both static and dynamic, appears to determine the pathway to product, although preorganization and selective binding of the final transition state structure in the multi-step carbocation cascade that forms epi</ ...[more]

PMID: 29308148

Similar Datasets

Reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi-isozizaene synthase.

Project description: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.

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Crystal structure of F95Q epi-isozizaene synthase, an engineered sesquiterpene cyclase that generates biofuel precursors ?- and ?-curcumene.

Project description:The saturated hydrocarbon bisabolane is a diesel fuel substitute that can be derived from sesquiterpene precursors bisabolene or curcumene. These sesquiterpenes are generated from farnesyl diphosphate in reactions catalyzed by eponymous terpenoid cyclases, but they can also be generated by engineered terpenoid cyclases in which cyclization cascades have been reprogrammed by mutagenesis. Here, we describe the X-ray crystal structure determination of F95Q epi-isozizaene synthase (EIZS), in which the new activity of curcumene biosynthesis has been introduced and the native activity of epi-isozizaene biosynthesis has been suppressed. F95Q EIZS generates ?- and ?-curcumene regioisomers with greater than 50% yield. Structural analysis of the closed active site conformation, stabilized by the binding of 3?Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, reveals a product-like active site contour that serves as the cyclization template. Remolding the active site contour to resemble curcumene instead of epi-isozizaene is the principal determinant of the reprogrammed cyclization cascade. Intriguingly, an ordered water molecule comprises part of the active site contour. This water molecule may also serve as a final proton acceptor, along with inorganic pyrophosphate, in the generation of curcumene regioisomers; it may also contribute to the formation of sesquiterpene alcohols identified as minor side products. Thus, the substitution of polar side chains for nonpolar side chains in terpenoid cyclase active sites can result in the stabilization of bound water molecules that, in turn, can serve template functions in isoprenoid cyclization reactions.

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Structure of epi-isozizaene synthase from Streptomyces coelicolor A3(2), a platform for new terpenoid cyclization templates.

Project description:The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 A resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with three Mg(2+) ions, inorganic pyrophosphate (PP(i)), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 A resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear to be well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-pi interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 A resolution crystal structure of F198A EIZS in a complex with three Mg(2+) ions, PP(i), and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS in a complex with four Hg(2+) ions has been determined at 1.90 A resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

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Characterization of a silent sesquiterpenoid biosynthetic pathway in Streptomyces avermitilis controlling epi-isozizaene albaflavenone biosynthesis and isolation of a new oxidized epi-isozizaene metabolite.

Project description:The genome-sequenced, Gram-positive bacterium Streptomyces avermitilis harbours an orthologue (SAV_3032) of the previously identified epi-isozizaene synthase (SCO5222) in Streptomyces coelicolor A3(2). The sav3032 is translationally coupled with the downstream sav3031 gene encoding the cytochrome P450 CYP170A2 analogous to SCO5223 (CYP170A1) of S. coelicolor A3(2), which exhibits a similar translation coupling. Streptomyces avermitilis did not produce epi-isozizaene or any of its oxidized derivatives, albaflavenols and albaflavenone, under in any culture conditions examined. Nonetheless, recombinant SAV_3032 protein expressed in Escherichia coli catalysed the Mg²+-dependent cyclization of farnesyl diphosphate to epi-isozizaene. To effect the production of epi-isozizaene in S. avermitilis, the sav3032 gene was cloned and placed under control of a copy of the native S. avermitilis promoter rpsJp (sav4925). The derived expression construct was introduced by transformation into a large-deletion mutant of S. avermitilis SUKA16 and the resulting transformants accumulated epi-isozizaene. The previously characterized oxidized epi-isozizaene metabolites (4R)- and (4S)-albaflavenols and albaflavenone, as well as a previously undescribed doubly oxidized epi-isozizaene derivative were isolated from cultures of S. avermitilis SUKA16 transformants in which sav3032 was coexpressed with the P450-encoding sav3031. This new metabolite was identified as 4β,5β-epoxy-2-epi-zizaan-6β-ol which is most likely formed by oxidation of (4S)-albaflavenol.

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An Aromatic Cluster in the Active Site of epi-Isozizaene Synthase Is an Electrostatic Toggle for Divergent Terpene Cyclization Pathways.

Project description:The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic precursor of the antibiotic albaflavenone. The hydrophobic active site is largely defined by aromatic residues that direct a multistep reaction sequence through multiple carbocation intermediates. The previous substitution of polar residues for a key aromatic residue, F96, converts EIZS into a high-fidelity sesquisabinene synthase: the F96S, F96M, and F96Q variants generate 78%, 91%, and 97% sesquisabinene A, respectively. Here, we report high-resolution X-ray crystal structures of two of these reprogrammed cyclases. The structures of the F96M EIZS-Mg2+3-risedronate and F96M EIZS-Mg2+3-inorganic pyrophosphate-benzyltriethylammonium cation complexes reveal structural changes in the F96 aromatic cluster that redirect the cyclization pathway leading from the bisabolyl carbocation intermediate in catalysis. The structure of the F96S EIZS-Mg2+3-neridronate complex reveals a partially occupied inhibitor and an enzyme active site caught in transition between open and closed states. Finally, three structures of wild-type EIZS complexed with the bisphosphonate inhibitors neridronate, pamidronate, and risedronate provide a foundation for understanding binding differences between wild-type and variant enzymes. These structures provide new insight regarding active site flexibility, particularly with regard to the potential for subtle expansion and contraction to accommodate ligands of varying sizes as well as bound water molecules. Additionally, these structures highlight the importance of conformational changes in the F96 aromatic cluster that could influence cation-π interactions with carbocation intermediates in catalysis.

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Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of epi-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes.

Project description:The sesquiterpene cyclase epi-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) Biochemistry 53, 1155-1168]. Here, we show that the substitution of hydrophobic residues-specifically, Y69, F95, F96, and W203-with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar-polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity.

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Biosynthesis of the sesquiterpene antibiotic albaflavenone in Streptomyces coelicolor. Mechanism and stereochemistry of the enzymatic formation of epi-isozizaene.

Project description:Epi-isozizaene synthase from Streptomyces coelicolor catalyzes the multistep cyclization of farnesyl diphosphate (2, FPP) to the tricyclic sesquiterpene hydrocarbon (+)-epi-isozizaene (3), which is converted in turn to the antibiotic albaflavenone (1) in a two-step, cytochrome P450-catalyzed oxidation. Competitive incubation of deuterated and nondeuterated samples of (3S)-NPP and (3RS)-NPP followed by GC-MS analysis of the degree of deuteration in the resulting labeled epi-isozizaene established that (3R)-NPP is the natural cyclization intermediate. Incubation of (3RS)-(Z)-[1-(2)H]NPP (4b) with epi-isozizaene synthase gave [11(anti)-(2)H]epi-isozizaene (3b), indicating that the S(N)' cyclization of 4 involves the predicted anti stereochemistry, consistent with the inference from earlier experiments with chirally deuterated FPP. Incubation of separate samples of [12,12,12-(2)H(3)]FPP (2d) and [13,13,13-(2)H(3)]FPP (2e) gave epi-isozizaenes 3d and 3e, thereby establishing the stereochemical course of the cyclization of the proposed intermediate acorenyl cation 6, as well as the stereochemistry of the successive 1,2-methyl migration and deprotonation that generate the final product. Further insights into the mechanism and the role of the enzyme came from site-directed mutagenesis of active site residues in two universally conserved Mg(2+)-binding domains and the identification of six minor sesquiterpene products 9-13 and 15 produced by the wild-type and mutant proteins. The aberrant products are believed to result from derailment and premature quenching of the normal intermediates of the cationic cyclization cascade.

| S-EPMC2690639 | biostudies-literature

Enhancing epi-cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi-cedrol synthase.

Project description:Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi-cedrol synthase (AaECS). The stop codon at the N-terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS-ECS produced sesquiterpene 8-epi-cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K m values for FPPS-ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi-cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi-cedrol in fermentation medium. The strain having fusion plasmid (pET32a-FPPS-ECS) produced 1.084 ± 0.09 mg/L epi-cedrol, while the strain harbouring mixed plasmid (pRSETB-FPPS and pET28a-ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I-TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N- C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.

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Zinc anode-compatible in-situ solid electrolyte interphase via cation solvation modulation.

Project description:The surface chemistry of solid electrolyte interphase is one of the critical factors that govern the cycling life of rechargeable batteries. However, this chemistry is less explored for zinc anodes, owing to their relatively high redox potential and limited choices in electrolyte. Here, we report the observation of a zinc fluoride-rich organic/inorganic hybrid solid electrolyte interphase on zinc anode, based on an acetamide-Zn(TFSI)2 eutectic electrolyte. A combination of experimental and modeling investigations reveals that the presence of anion-complexing zinc species with markedly lowered decomposition energies contributes to the in situ formation of an interphase. The as-protected anode enables reversible (~100% Coulombic efficiency) and dendrite-free zinc plating/stripping even at high areal capacities (>2.5?mAh?cm?2), endowed by the fast ion migration coupled with high mechanical strength of the protective interphase. With this interphasial design the assembled zinc batteries exhibit excellent cycling stability with negligible capacity loss at both low and high rates.

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Modulation of brain cation-Cl- cotransport via the SPAK kinase inhibitor ZT-1a.

Project description:The SLC12A cation-Cl- cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

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