Probing the reaction mechanism of aristolochene synthase with 12,13-difluorofarnesyl diphosphate

Yu, Fanglei; Miller, David J.; Allemann, Rudolf Konrad

doi:10.1039/b709562g

Cited by 29 publications

(32 citation statements)

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“…Section 1734 solely to indicate this fact. with the intact substrates FPP and 2-fluorofarnesyl diphosphate (2F-FPP) (12), and the inhibitor 12,13-difluorofarnesyl diphosphate (DF-FPP) (13). Differences observed in active site conformations and substrate conformations appear to be linked to differences in metal binding, analysis of which suggests a possible sequence for metal ion binding and conformational changes required for catalysis.…”

Section: Farnesyl Diphosphate (Fpp)mentioning

confidence: 99%

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X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

Shishova

Miller

et al. 2008

Journal of Biological Chemistry

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Farnesyl diphosphate (FPP),2 a flexible 15-carbon isoprenoid, is the universal precursor of Ͼ300 different cyclic sesquiterpenes found in numerous plants, bacteria, and fungi (1, 2). The cyclization of FPP is catalyzed by a sesquiterpene cyclase that utilizes a trinuclear magnesium cluster to trigger the departure of the pyrophosphate (PP i ) leaving group, thereby forming an allylic carbocation that typically reacts with one of the remaining bonds of the substrate (3-7). The remarkable diversity of sesquiterpene structure and stereochemistry is the consequence of precise control exerted by the cyclase over the conformations of the flexible substrate and carbocation intermediates in the cyclization cascade.Aristolochene synthase from Aspergillus terreus is a sesquiterpene cyclase that catalyzes the cyclization of FPP to form aristolochene (Fig. 1a), the parent hydrocarbon of a large group of fungal toxins such as gigantenone, PR-toxin, and bipolaroxin (8). In contrast to aristolochene synthase from Penicillium roqueforti, which generates aristolochene predominantly (Ͼ90%) but also small amounts of germacrene A and valencene (9, 10), aristolochene synthase from A. terreus generates aristolochene exclusively (9). Each cyclase adopts the common ␣-helical fold of a class I terpenoid cyclase and contains two conserved metal binding motifs: the "aspartate-rich" motif D 90 DLLE that coordinates to Mg 3 ⅐PP i stabilizes the active site in a closed conformation that is completely sequestered from bulk solvent (Fig. 1b) (11). In addition to multiple metal coordination interactions, the PP i anion accepts hydrogen bonds from conserved residues Arg , and Tyr 315 when bound to the closed conformation (Fig. 1c). It is likely that the diphosphate group of FPP makes identical metal coordination and hydrogen bond interactions in the Michaelis complex, i.e. the complex between the enzyme and the productively bound substrate that immediately precedes the initiation of the cyclization cascade.Substrate conformation is a crucial determinant of the biosynthetic outcome of the terpenoid cyclase reaction. The active site of aristolochene synthase from A. terreus serves as a high fidelity template that fixes FPP in a single, productive conformation in the Michaelis complex; otherwise, aberrant cyclization products would result. To study the conformational control of FPP in the active site of aristolochene synthase from A. terreus, we now report the structures of crystalline complexes * This work was supported, in whole or in part, by National Institutes of Health Grants GM 56838 (to D. W. C.), GM 30301 (to D. E. C.), and GM 13956 (to R. M. C.). This work was also supported by the Engineering and Physical Sciences Research Council (Grant EP/D069580 to R. K. A.) and by Cardiff University (Studentship to F. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Th...

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Section: Farnesyl Diphosphate (Fpp)mentioning

confidence: 99%

“…The syntheses of 2F-FPP and DF-FPP have been reported (12)(13)(14), and an additional synthesis of 2F-FPP is outlined in the supplemental materials. Recombinant A. terreus aristolochene synthase was expressed in Escherichia coli BL21(DE3)pLysS and purified as previously described (11,15).…”

Section: Enzyme Incubations With 2f-fpp and Df-fpp In Solution-mentioning

confidence: 99%

X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

Shishova

Miller

et al. 2008

Journal of Biological Chemistry

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“…[5] Notably, Virgil and Corey demonstrated that lanosterol synthase could utilize vinyl ether 1 as a substrate in place of squalene oxide. [5a] In subsequent work by Jin and Coates, [5b] vinyl methyl ether-containing isoprenoid diphosphate 2 was used as a substrate for a diterpene synthase. This vinyl methyl ether, however, hydrolyzed rapidly in neutral aqueous conditions.…”

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High‐Throughput Screening for Terpene‐Synthase‐Cyclization Activity and Directed Evolution of a Terpene Synthase

et al. 2013

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A synthetic substrate enables a new colorimetric screen for terpene synthase cyclization activity, facilitating the engineering of these enzymes. Using directed evolution, the thermostability of the sesquiterpene synthase BcBOT2 was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase, SSCG_02150.

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“…,22 10 mM IPP, and 10 mM MgCl2 were incubated with 5 µM FPPS in 450 µL of 20 mM Tris buffer, pH 7.9, containing 500 mM NaCl overnight at RT, and the crude reaction mixture then stored at -20 °C.The thawed sample was filtered by centrifugation through an Amicon Ultra filter with 10 kDa molecular mass cutoff, and the flow-through fraction collected and lyophilized to dryness. After re-suspension of the lyophilized powder in 500 µL of D2O, and recording of a 1 H NMR spectrum, the presence of 3 vinyl protons matched a previously reported spectrum22 of 12,13-DFFPP and indicated that the enzymatic elongation of 8,9-DFGPP to 12,13-DFFPP had been successful. The sample was then lyophilized, re-suspended in 50 µL H2O, and stored at -20 °C until use.…”

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Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Matos

Kumar

Alison

et al. 2020

Preprint

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Accession CodesThe atomic coordinates and structure factors for the DFFPP-PS complex have been deposited in the Protein Data Bank. RCSB PDB entry 6WKC (WT-Mg), 6WKD (WT-DFFPP), 6WKE DFFPP) for the respective models. ABBREVIATIONS FPP, farnesyl diphosphate; DFFPP, 12,13-difluorofarnesyl diphosphate; PS, pentalenene synthase, CS, caryolan-1-ol synthase. SUPPORTING INFORMATION AVAILABLE AbstractMost terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated.In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-p interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76.Mutagenesis experiments also support a role for residue 76 in cation-p interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

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Probing the reaction mechanism of aristolochene synthase with 12,13-difluorofarnesyl diphosphate

Abstract: 12,13-Difluorofarnesyl diphosphate, prepared using Suzuki-Miyaura chemistry, is a potent inhibitor of aristolochene synthase (AS), indicating that the initial cyclisation during AS catalysis generates germacryl cation in a concerted reaction.

Cited by 29 publications

References 22 publications

X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

X-ray Crystallographic Studies of Substrate Binding to Aristolochene Synthase Suggest a Metal Ion Binding Sequence for Catalysis

High‐Throughput Screening for Terpene‐Synthase‐Cyclization Activity and Directed Evolution of a Terpene Synthase

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

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