Sesquiterpene Synthases Cop4 and Cop6 from Coprinus cinereus: Catalytic Promiscuity and Cyclization of Farnesyl Pyrophosphate Geometric Isomers

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 capable of isomerizing the C2-C3 π bond of all-trans-FPP. Cop6 is a “high-fidelity” α-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)-β-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)-β-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 may explain their different catalytic fidelities.

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

“…S2 for mass spectra of identified sesquiterpenoids, and Table S3 for in vitro product profiles with isolated enzymes in the supplemental material). As reported previously for the wild-type enzymes (2,3,22), Cop4 makes 10 different sesquiterpene products with ␦-cadinene 8 (compounds are numbered; see Fig. 2 and 4 for structures) and ␤-copaene 6 as the major products in vivo, whereas Cop6 makes almost exclusively (Ϫ)-␣-cuprenene 1.…”

“…All cloning and investigations of sesquiterpene biosynthesis were carried out in Escherichia coli strain JM109 or E. coli BL21. Plasmids pUC-Cop3, pET21-Cop4, and pHis8-Cop6 (Cop6 and Cop4 contain a His 8 tag and a His 6 tag, respectively, added to their N termini for affinity purification) for overexpression of sesquiterpene synthases from Coprinus cinereus in E. coli have been described previously (2,3,22) (see Table S1 in the supplemental material). E. coli cultures were grown at 30°C and 250 rpm in Luria-Bertani (LB) medium supplemented with 100 g ml Ϫ1 of the appropriate antibiotics (ampicillin for pUC-Cop3 and pET21-Cop4, kanamycin for pHis8-Cop6).…”

“…For kinetic measurements, Cop4 and Cop6 wild-type and loop mutants were overexpressed as described above, and the histidine-tagged recombinant proteins were purified by affinity chromatography as described previously (2,3,22). Protein concentrations were determined by using Bradford reagent (Bio-Rad, Hercules, CA).…”

“…The four closely related enzymes (Cop1 to Cop4) each synthesized multiple cyclization products, while trichodiene synthase homolog Cop6 was highly specific. In a subsequent study we characterized the cyclization pathways of Cop4 and Cop6 and compared homology models of the two enzymes, indicating that different binding pocket sizes may be responsible for their different catalytic fidelities by allowing FPP to bind in one or several conformations (22). Cop4 rearranges a secondary cisoid, allylic cation derived from FPP after isomerization (cis-trans pathway of catalysis) into multiple products, with ␦-cadinene and ␤-copaene as the major products when expressed in E. coli and germacrene D and cubebol as the major products in vitro.…”

“…Retention indices and mass spectra of compound peaks were compared to reference data in Mass Finder's (software version 3) terpene library (19). In addition, essential oils with known terpene compositions were used as authentic standards for the major products as previously described (2,3,22).…”