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. 2010 May 17;11(8):1093-106.
doi: 10.1002/cbic.200900671.

Sesquiterpene synthases Cop4 and Cop6 from Coprinus cinereus: catalytic promiscuity and cyclization of farnesyl pyrophosphate geometric isomers

Fernando Lopez-Gallego  1 , Sean A Agger, Daniel Abate-Pella, Mark D Distefano, Claudia Schmidt-Dannert
Affiliations

Sesquiterpene synthases Cop4 and Cop6 from Coprinus cinereus: catalytic promiscuity and cyclization of farnesyl pyrophosphate geometric isomers

Fernando Lopez-Gallego et al. Chembiochem. .

Abstract

Sesquiterpene synthases catalyze with different catalytic fidelity the cyclization of farnesyl pyrophosphate (FPP) into hundreds of known compounds with diverse structures and stereochemistries. Two sesquiterpene synthases, Cop4 and Cop6, were previously isolated from Coprinus cinereus as part of a fungal genome survey. This study investigates the reaction mechanism and catalytic fidelity of the two enzymes. Cyclization of all-trans-FPP ((E,E)-FPP) was compared to the cyclization of the cis-trans isomer of FPP ((Z,E)-FPP) as a surrogate for the secondary cisoid neryl cation intermediate generated by sesquiterpene synthases, which are capable of isomerizing the C2--C3 pi bond of all-trans-FPP. Cop6 is a "high-fidelity" alpha-cuprenene synthase that retains its fidelity under various conditions tested. Cop4 is a catalytically promiscuous enzyme that cyclizes (E,E)-FPP into multiple products, including (-)-germacrene D and cubebol. Changing the pH of the reaction drastically alters the fidelity of Cop4 and makes it a highly selective enzyme. Cyclization of (Z,E)-FPP by Cop4 and Cop6 yields products that are very different from those obtained with (E,E)-FPP. Conversion of (E,E)-FPP proceeds via a (6R)-beta-bisabolyl carbocation in the case of Cop6 and an (E,E)-germacradienyl carbocation in the case of Cop4. However, (Z,E)-FPP is cyclized via a (6S)-beta-bisabolene carbocation by both enzymes. Structural modeling suggests that differences in the active site and the loop that covers the active site of the two enzymes might explain their different catalytic fidelities.

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Figures

Figure 1
Figure 1. Stereochemical analysis of β-bisabolene products synthesized from (Z,E)-FPP
Reaction products of purified Cop4 (A) and mutant Cop6 N224D (B) with (Z,E)-FPP were separated by chiral GC-MS (dotted trace) and identified using authentic β–bisabolene standards (solid trace). Peaks labeled with an asterisk correspond to the Cop4 and Cop6 β-bisabolene reaction products.
Figure 2
Figure 2. Effect of pH on the product selectivity of Cop4 with (E,E)-FPP
Reactions of purified Cop4 with (E,E)-FPP were carried under different pH conditions. Reaction products were separated by GC-MS and compound peaks identified by comparison of mass spectra and RI values with those in reference libraries and with authentic standards.
Figure 3
Figure 3. Structural modeling of Cop4 and Cop6 based on the structures of aristolochene synthase (ACH) [20] and trichodiene synthase (TCH) [48], respectively
Top panels: Hydrogen bond and metal coordination interactions in the enzyme-Mg2+-PPi-complex for the Cop6 (A) (green side chains) and Cop4 (B) (orange side chains) models. Depicted side chains of the conserved DDXXD/E, NSE/DTE and basic motif (XRY) are superimposed with corresponding side chains from their respective template structures (TCH, purple; ACH, yellow). Mg2+ ions are shown in green and complexed PPi is located in the center of the networks. Center panels: View into the active site cavities of unliganded Cop6 (C) (green alpha carbons) and Cop4 (D) (orange alpha carbons). Positively charged and negatively charged amino acid residues are shown in blue and red respectively. Bottom panel: Superimposition of the unliganded Cop4 and Cop6 models with the unliganded structures of THC and ACH (E). Loops covering the active sites of the enzymes are labeled and colors correspond to those of their alpha carbon backbones in the superimposition. Inset shows the amino acid residues of the different loops. Basic (blue) and acidic (red) amino acid residues are highlighted.
Scheme 1
Scheme 1. Proposed reaction mechanism accounting for the identified products generated from (E,E)-FPP by Cop4, Cop6 and NS1
Numbered reaction arrows indicate different branch points in the cyclization reaction. Relative amounts of products formed by each enzyme are shown in Table 2.
Scheme 2
Scheme 2. Proposed reaction mechanism accounting for the identified products generated from (Z,E)-FPP by Cop4, Cop6 and NS1
Numbered reaction arrows indicate different branch points in the cyclization reaction. Relative amounts of products formed by each enzyme are shown in Table 2. (6S)-β-bisabolene is also formed as a product (18%) by the mutant form of Cop 6 (N224D), thus providing strong evidence for the common 6S stereochemistry of β-bisabolyl cation intermediate generated by Cop 6.
Scheme 3
Scheme 3. Postulated mechanism of monoterpene formation from E-GPP
Acylic monoterpenes produced by NS1 are proposed to be derived from a transoid geranyl cation, while the acyclic and cyclic monoterpene products of Cop4 and Cop6 are postulated to involve the generation of a cisoid geranyl cation (neryl cation). Relative amounts of products formed by each enzyme are shown in Table 2.
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