Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of <i>epi</i>-Isozizaene Synthase

Li, Ruiqiong; Chou, Wayne; Himmelberger, Julie A.; Litwin, K.; Harris, Golda G.; Cane, David E.; Christianson, D.W.

doi:10.1021/bi401643u

Cited by 64 publications

(138 citation statements)

References 47 publications

(119 reference statements)

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“…26 The formation of alternative products is a consequence of remolding the hydrophobic active site contour, which then redirects the cyclization cascade. Curiously, the F96Y and F198Y substitutions did not deactivate the enzyme – a newly introduced tyrosine hydroxyl group could be susceptible to alkylation by a carbocation intermediate in catalysis, but the hydrophobic active site nevertheless tolerated this substitution to yield a functional catalyst.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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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 8 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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Section: Introductionmentioning

confidence: 99%

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

et al. 2017

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“…1). However, structural data and mechanistic considerations explaining this promiscuity on the molecular level are very limited (8,(21)(22)(23)(24)(25). The role of the protein scaffold has been widely thought to chaperone the cyclization cascade by merely conformational control (22,26).…”

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Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

Schrepfer

Buettner

Goerner

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Class I terpene synthases generate the structural core of bioactive terpenoids. Deciphering structure-function relationships in the reactive closed complex and targeted engineering is hampered by highly dynamic carbocation rearrangements during catalysis. Available crystal structures, however, represent the open, catalytically inactive form or harbor nonproductive substrate analogs. Here, we present a catalytically relevant, closed conformation of taxadiene synthase (TXS), the model class I terpene synthase, which simulates the initial catalytic time point. In silico modeling of subsequent catalytic steps allowed unprecedented insights into the dynamic reaction cascades and promiscuity mechanisms of class I terpene synthases. This generally applicable methodology enables the active-site localization of carbocations and demonstrates the presence of an active-site base motif and its dominating role during catalysis. It additionally allowed in silico-designed targeted protein engineering that unlocked the path to alternate monocyclic and bicyclic synthons representing the basis of a myriad of bioactive terpenoids.computational biology | closed complex modeling | protein engineering | terpene synthases | terpene synthase catalysis

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“…Promiscuity in these enzymes is not surprising given the multiple cyclization and rearrangement steps that the substrates often undergo during catalysis, any one of which can be altered by perturbations to the microenvironment of the active site. Promiscuity is often seen in mutagenesis experiments where a few key mutations are typically sufficient to alter the product selectivity of the enzymes (17,18). As a result, prediction of function for terpene synthases is still a very challenging task in the field of computational biology.…”

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confidence: 99%

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

Chow

Tian

Ramamoorthy

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.T erpenoid compounds form a large group of natural products synthesized by many species of plants, fungi, and bacteria. To date, more than 70,000 of these compounds have been identified, comprising ∼25% of all of the known natural products (Dictionary of Natural Products database, dnp.chemnetbase.com/intro/ index.jsp). Terpenoids and molecules containing terpenoid fragments are produced from two basic five-carbon building blocks: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (1, 2). IPP and DMAPP are then converted into a series of terpenoid diphosphate esters of increasing chain lengths by chain length selective polyprenyl diphosphate synthases to give geranyl diphosphate (GPP, C 10 ), farnesyl diphosphate (FPP, C 15 ), geranylgeranyl diphosphate (GGPP, C 20 ), and higher five-carbon homologs (3). These molecules are substrates for terpene synthases, which catalyze hydroxylation, elimination, cyclization, and rearrangement reactions to produce much of the structural diversity of terpenoid molecules found in nature (4, 5).The two main classes of terpene synthases (class I and class II) are distinguished from one another by the mechanisms they use to initiate carbocationic cyclization and rearrangement reactions and by their respective folds (6, 7). Class I terpene synthases use active site Mg 2+ ions to bind and activate the diphosphate moieties of their substrates as leaving groups to generate highly reactive allylic carbocations. In those terpene synthases that catalyze cyclization reactions, the carbocations alkylate distal double bonds in the hydrocarbon chain. The initial cycliza...

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Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

Cited by 64 publications

References 47 publications

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

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

Identification of amino acid networks governing catalysis in the closed complex of class I terpene synthases

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus

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