Biosynthesis of the Sesquiterpene Botrydial in <i>Botrytis cinerea</i>. Mechanism and Stereochemistry of the Enzymatic Formation of Presilphiperfolan-8β-ol

Wang, Chieh-Mei; Hopson, Russell; Lin, Xin; Cane, David E.

doi:10.1021/ja9021649

Cited by 94 publications

(93 citation statements)

References 19 publications

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“…For example, presilphiperfolan-8β-ol synthase catalyzes the highly selective seven-step transformation of FPP to presilphiperfolan-8β-ol (39,43). Each of these steps is exquisitely sensitive to the steric and electrostatic environment of the active site, and single mutations are often sufficient to substantially alter the structures of the products.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…Alignment of functionally characterized fungal sesquiterpene synthase sequences shows that C. cinereus sesquiterpene synthases (2,3,22), longifolene synthase from Fusarium graminearum (23), and presilphiperfolan-8-␤-ol synthase from Botrytis cinerea (26,38), but not aristolochene synthase AtACS (30) and trichodiene synthase FsTHS (27), contain a conserved Asn in the H-␣1 loop (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

López‐Gallego

Wawrzyn

Schmidt‐Dannert

2010

Appl Environ Microbiol

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Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-␣1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-␣1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-␣1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-␣1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-␣1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.Sesquiterpene synthases catalyze the cyclization of farnesyl pyrophosphate (FPP) to structurally diverse C 15 -hydrocarbons. These enzymes belong to the large group of terpene synthases that convert isoprene pyrophosphate substrates into hundreds of described terpenoid compounds by employing some of the most complex carbon-carbon forming reactions known (8). Many terpenoids are biologically active and are produced by plants, bacteria, and fungi such as, for example, antibiotics, toxins, and pheromones (5, 9).Catalysis in this class of enzymes is dependent on the presence of three Mg 2ϩ ions coordinated by two conserved motifs, an aspartate-rich DDXXD/E and an NSE/DTE motif, flanking the entrance of the active site. This Mg 2ϩ cluster binds the pyrophosphate (PP i ) group of FPP and positions the isoprenyl chain in the hydrophobic substrate binding pocket of the enzyme (10). Substrate binding triggers a conformational change that results in the closure of the active site and concurrent PP i cleavage to generate an initial transoid, allylic carbocation (6,7,35). This carbocation is then transferred along the isoprenyl chain and eventually quenched either by a water molecule or through proton abstraction. The binding pocket determines folding of the isoprenyl chain and chaperones the reactive carbocation intermediates until the final quenching step (20), thereby defining the product profile of a particular sesquiterpene synthase.Crystal structures have been solved for several microbial and plant sesquiterpene synthases (1,7,14,30). All enzymes share the same ␣-helical fold characteristic for ionization-dependent terpene synthases. Plant enzymes possess an additional, catalytically inactive N-terminal domain that h...

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“…[15][16][17][18][19] Recently, the presilphiperfolan-8b-ol synthase from Botrytis cinerea was described. [20,21] Here we present the identification of two terpene cyclases from F. fujikuroi, the construction of genetically engineered strains for optimized terpene production, and the isolation and structural elucidation of the enzymes' main products.…”

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confidence: 99%

Genetic Dissection of Sesquiterpene Biosynthesis by Fusarium fujikuroi

et al. 2013

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Biosynthesis of the Sesquiterpene Botrydial in Botrytis cinerea. Mechanism and Stereochemistry of the Enzymatic Formation of Presilphiperfolan-8β-ol

Cited by 94 publications

References 19 publications

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

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

Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

Genetic Dissection of Sesquiterpene Biosynthesis by Fusarium fujikuroi

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