Stereochemistry and deuterium isotope effects associated with the cyclization-rearrangements catalyzed by tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases, and the chimeric CH₄ hybrid cyclase

Abstract

Tobacco epiaristolochene and hyoscyamus premnaspirodiene synthases (TEAS³ and HPS) [1,2] are terpene cyclases responsible for the production of the respective sesquiterpene hydrocarbons (2 and 5) in the biosynthesis of the oxygenated phytoalexins capsidiol (4), solavetivone (6), lubimin (7), rishitin (8), and related metabolites in sweet pepper, tobacco, potato, and other Solanaceae (Scheme 1) [3–5].

Scheme 1

Biosynthesis of the sesquiterpene phytoalexins capsidiol (4), solavetivone (6), lubimin (7), and rishitin (8) from (E,E)-farnesyl diphosphate (1) by way of epiaristolochene (2) and premnaspirodiene (5).

Incorporation of [1,2-^C₂]acetate into capsidiol in Capsicum annum cultures and NMR analyses confirmed the occurrence of a methyl migration in the biosynthesis of this eremophilane sesquiterpene [6]. Retention of deuterium at C4 of capsidiol biosynthesized from [4,4-^H₂]mevalonate demonstrated that the unusual trans-stereochemistry of the vicinal methyl groups could not be attributed to epimerization of a cis-eremophilane precursor [7].

The hydrocarbon intermediate isolated from elicitor-treated cell cultures of Nicotiana tobacum [8] was identified as (+)-epiaristolochene (2) by comparison with the diene prepared chemically by deoxygenation of capsidiol [9], the structure and stereochemistry of which were established by an X-ray crystallo-graphic analysis [10]. Kinetic analysis of the oxidation of 2 to capsidiol and the potential intermediates epiaristolochen-1β- and -3α-ol with the cytochrome P₄₅₀ oxidase epiaristolochene dihydroxylase (EAH) established the preferred order of the oxidation steps, 2 → 3 → 4 [11,12]. The sequence of oxidative transformations and intermediates in the conversion of solavetivone to lubimin and rishitin was elucidated by incorporation of deuterium and carbon-13 labeled precursors [13–15]. The presumed sesquiterpene precursor (−)-premnaspirodiene (5) [16] has been synthesized as the pure enantiomer [17] and in racemic form [18], and its identification as the product of HPS rests primarily on GC–MS evidence [19].

The usual mechanism for the closely related processes catalyzed by TEAS and HPS are presented in Scheme 2 [1,2]. Intramolecular alkylation of the terminal double bond of the FPP substrate generates the enzyme bound gremacrene A intermediate (9). Proton-induced cyclization of 9 in a boat-chair conformation leads to a 5,10-syn eudesmyl ion 10A that undergoes 5 → 4 hydride shift to form the branch point intermediate 10B. 1,2-Methyl migration followed by proton elimination give rise to epiaristolochene (2) and epieremophilene (13), whereas ring contraction by C-9 methylene rearrangement and elimination produces premnaspirodiene (5).

Scheme 2

Proposed mechanisms for the cyclizations of (E,E)-farnesyl diphosphate (1) to epiaristolochene (2), epieremophilene (13), and premnaspirodiene (5) catalyzed by the respective synthases TEAS, HPS, and the CH₄ hybrid. The reactions proceed through germacrene A (9), and isomeric eudesmyl (10A and 10B), epi-eremophilenyl (11), and vetispirenyl (12) carbocation intermediates. In the case of TEAS, angular methyl migration is favored along the upper branch to the fused ring product whereas in the case of HPS ring methylene rearrangement on the opposite face and proton elimination at C1 lead to the spiro[5.4]decane product. The CH₄ mutant form affords a mixture of both products, together with the double bond isomer epieremophilene (13).

The cloning and over-expression of recombinant TEAS and HPS in Escherichia coli provided sufficient quantities of these interesting cyclases for structural and mechanistic investigations [20]. These two proteins are 77% identical and 81% similar based on amino acid sequence alignment, contain the familiar DDXXD aspartate triad linked to diphosphate binding, and have kinetic characteristics typical of terpene cyclases. One of a series of chimeric proteins resulting from domain swapping designated CH₄ catalyzed the conversion of 1 to epiaristolochene, premnaspirodiene, and an unknown product subsequently identified as epieremophilene (13), i.e., the Δ ^{isomer of} 2 [11,21]. Pre-steady state kinetic data are consistent with either a rate-determining cyclization to the germacrene A intermediate or final dissociation of the bicyclic products, and the existence of a single catalytic site on the CH₄ hybrid enzyme [22]. X-ray diffraction analysis of crystalline TEAS in the absence and presence of inert substrate mimics revealed a hydrophobic, aromatic-rich binding pocket in the C-terminal domain with twoMg ^{ions positioned near the opening [}23]. The production of germacrene A (9) by the point mutant TEAS-Y520-F confirmed the identity of the macrocyclic intermediate and a role for tyrosine 520 in catalysis, perhaps in contributing to an H-bonding network making up the back of the active site pocket [24].

The functionally similar aristolochene synthases isolated from the fungi Aspergillus terreus and Penicillum roquaforti catalyze the conversion of FPP to the 4β-CH₃, 7α-isopropenyl diastereomer of 2 [1,2]. However, the amino acid sequences of these fungal cyclases differ appreciably from those of TEAS and HPS. Labeling experiments established that the macrocyclization step occurs with inversion at C-1 of the substrate and proton elimination at the cis-terminal methyl position, and that conversion of the enzyme-bound intermediate to aristolochene takes place with 10 → 5 methyl migration and syn-related proton elimination [25,26]. Indirect evidence supporting the same germacrene A intermediate (9) was obtained by enzyme-catalyzed cyclization of (7R)-6,7-dihydro FPP to 6,7-dihydro germacrene A [27].

The objectives of the present work were to analyze the timing of the cyclization steps and the stereochemistry of the individual protonation and deprotonation events in the mechanism by means of deuterium labeling. In this paper, we report syntheses of farnesyl PPs bearing deuterium in the terminal methyl groups and in the pro-R and pro-S positions at C-8. The stereochemistry of proton transfer steps was elucidated by GC–MS and/or NMR analyses of the products from incubations of the labeled substrates with the three enzymes and from incubations conducted in D₂O. Comparisons of the isotope effects and stereospecificities of the different reactions catalyzed by TEAS, HPS, and CH₄ afford new insights on the positions of active site acids and bases, on the determinants of product specificity, and on the rate determining steps.

Footnotes