Studies of the cryptic allylic pyrophosphate isomerase activity of trichodiene synthase using the anomalous substrate 6,7-dihydrofarnesyl pyrophosphate

Abstract

Two enantiomeric analogues of farnesyl pyrophosphate (1) were tested as inhibitors and anomalous substrates of trichodiene synthase, which catalyzes the cyclization of trans,trans-farnesyl pyrophosphate (1) to the sesquiterpene hydrocarbon trichodiene (2). The reaction has been shown to involve preliminary isomerization of 1 to the tertiary allylic isomer nerolidyl pyrophosphate (3) which is cyclized without detectable release of the intermediate from the active site of the cyclase. Both (7S)-trans-6,7-dihydrofarnesyl pyrophosphate (7a) and (7R)-trans-6,7-dihydrofarnesyl pyrophosphate (7b), prepared from (3R)- and (3S)- citronellol (9a and 9b), respectively, proved to be modest competitive inhibitors of trichodiene synthase. The values of Ki(7a), 395 nM, and Ki(7b), 220 nM, were 10-15 times the observed Km for 1 and half the Ki of inorganic pyrophosphate alone. Incubation of either 7a or 7b with trichodiene synthase resulted in formation of a mixture of products which by radio/gas-liquid chromatographic and GC/selected ion mass spectrometric analysis was shown to be composed of 80-85% isomeric trienes 19-21 and 15-20% allylic alcohols 12 and 18. Examination of the water-soluble products resulting from incubation of 7a also revealed the generation of 24% of the isomeric cis-6,7-dihydrofarnesyl pyrophosphate (26). The combined rate of formation of anomalous alcoholic and olefinic products was 10% the Vmax determined for the conversion of 1 to 2. The results can be explained by initial enzyme-catalyzed isomerization of dihydrofarnesyl pyrophosphate (7) to the corresponding tertiary allylic isomer dihydronerolidyl pyrophosphate (8). Since the latter intermediate is unable to cyclize due to the absence of the 6,7-double bond, ionization of 8 and quenching of the resulting ion pair by deprotonation, capture of water, or collapse to the isomeric primary pyrophosphate esters will generate the observed spectrum of anomalous products.

Structural and Chemical Biology of Terpenoid Cyclases

“… 257 Notably, incubation of trichodiene synthase with the reduced substrate analogue 7( S )- trans -6,7-dihydrofarnesyl diphosphate or its enantiomer yields product arrays that reflect formation of the corresponding 6,7-dihydronerolidyl diphosphate intermediate, thereby providing further support for 3( R )-nerolidyl diphosphate as an intermediate in the cyclization cascade. 258 …”

“…Enzymological studies using isotopically labeled FPP and nerolidyl diphosphate confirm that 3­( R )-nerolidyl diphosphate is a catalytic intermediate, as expected for the ionization-recombination-reionization sequence that enables initial C1–C6 bond formation. , Single-turnover experiments indicate that FPP ionization is the rate-determining chemical step of catalysis, with product release being the overall rate-determining step of catalysis . Notably, incubation of trichodiene synthase with the reduced substrate analogue 7­( S )- trans -6,7-dihydrofarnesyl diphosphate or its enantiomer yields product arrays that reflect formation of the corresponding 6,7-dihydronerolidyl diphosphate intermediate, thereby providing further support for 3­( R )-nerolidyl diphosphate as an intermediate in the cyclization cascade …”

The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene

“… By comparison of the homology models for ADS and BFS, we chose to mutate T296 of ADS to the nearly isosteric but less polar V296, corresponding to amino acid residue V324 in BFS. This amino acid residue is located on the wall of the active site cavity just left below D299 of ADS (Figure ), the first amino acid of the universally conserved aspartate-rich DDxxD motif, which has been implicated in binding of essential divalent Mg 2+ ions that are in turn coordinated to the pyrophosphate moiety of the FPP substrate. ,− Incubation of this T296V mutant with ( E , E )-FPP resulted in a major change in product distribution that at that point consisted of 88.5% ( E )-β-farnesene, 7.4% amorpha-4,11-diene, and a mixture of minor products (4%) (Figure , Figure S2, and Table ).…”

“…Sesquiterpene synthases convert the universal acyclic precursor (2 E ,6 E )-farnesyl diphosphate [( E , E )-FPP] into more than 300 types of acyclic, monocylic, bicyclic, and tricyclic sesquiterpenes. − Although the cyclization mechanisms have been extensively studied, and the structures of several sesquiterpene synthases of bacterial, fungal, and plant origin have been determined, − knowledge of the precise protein structural factors that control the initiation of the cyclization cascade and the resultant product structure is still limited.…”

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

“…Examples of trans-pathway specific enzymes with solved crystal structures include pentalenene synthase form Streptomyces UC5319 [16], 5-epi-aristolochene synthase from Nicotiana tobaccum [17, 18] and aristolochene synthases from Aspergillus terreus and Penicillium roqueforti [19, 20]. Trichodiene synthase from F. sporotrichoides [21, 22] and very recently, δ-cadinene synthase from Gossypium arboreum (cotton) [23] are the only cis-trans pathway specific sesquiterpene synthase with a solved crystal structure. Examples for catalytically very promiscuous sesquiterpene synthases are γ-humulene synthase and δ-selinene synthase from grand fir .…”