The α-Terpineol to 1,8-Cineole Cyclization Reaction of Tobacco Terpene Synthases

Abstract

Flowers of Nicotiana species emit a characteristic blend including the cineole cassette monoterpenes. This set of terpenes is synthesized by multiproduct enzymes, with either 1,8-cineole or a-terpineol contributing most to the volatile spectrum, thus referring to cineole or terpineol synthase, respectively. To understand the molecular and structural requirements of the enzymes that favor the biochemical formation of a-terpineol and 1,8-cineole, site-directed mutagenesis, in silico modeling, and semiempiric calculations were performed. Our results indicate the formation of a-terpineol by a nucleophilic attack of water. During this attack, the a-terpinyl cation is stabilized by p-stacking with a tryptophan side chain (tryptophan-253). The hypothesized catalytic mechanism of a-terpineol-to-1,8-cineole conversion is initiated by a catalytic dyad (histidine-502 and glutamate-249), acting as a base, and a threonine (threonine-278) providing the subsequent rearrangement from terpineol to cineol by catalyzing the autoprotonation of (S)-(2)-a-terpineol, which is the favored enantiomer product of the recombinant enzymes. Furthermore, by site-directed mutagenesis, we were able to identify amino acids at positions 147, 148, and 266 that determine the different terpineol-cineole ratios in Nicotiana suaveolens cineole synthase and Nicotiana langsdorffii terpineol synthase. Since amino acid 266 is more than 10 Å away from the active site, an indirect effect of this amino acid exchange on the catalysis is discussed.

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

“…It has been demonstrated that other plant CinSs also accumulate alternative products at significant amounts when expressed in the monoterpenoid production platform. For example, CinS from A. thaliana produces only 42 % cineole with α‐terpineol (19 %), β‐myrcene (16 %), and sabinene (14 %) making up the majority of the rest of the product profile . The question arises, how is bCinS able to tightly control the carbocation intermediates without leakage to other products and/or premature quenching?…”

“…Protonation of the other carbon of the double bond is highly unfavourable, as this would lead to a secondary, rather than tertiary, carbocation, and in our model calculations this spontaneously rearranges to the tertiary cation. The proton acceptor for the final step is unknown, and there is no obvious proton relay network present like the one proposed for CinS from Nicotiana forgetiana , where the hydroxy group of Tyr496 or Thr278 could act as proton acceptor for deprotonation of ( R )‐(+)‐α‐terpineol or ( S )‐(−)‐α‐terpineol, respectively . The most likely candidate for proton abstraction in bCinS is Asn305, due to its close proximity to C7 and strong interaction with the water molecule during simulation.…”

“…For example, CinS from A. thaliana produces only 42 % cineole with a-terpineol (19 %), b-myrcene (16 %), and sabinene (14 %) making up the majority of the rest of the product profile. [13,16] The question arises, how is bCinS able to tightly control the carbocation intermediates without leakage to other products and/or premature quenching? Also, what (stereochemical) intermediates are formed during the cyclisation cascade?…”

“…The proton acceptor for the final step is unknown, and there is no obvious proton relay network present like the one proposed for CinS from Nicotiana forgetiana, where the hydroxy group of Tyr496 or Thr278 could act as proton acceptor for deprotonation of (R)-(+)-a-terpineol or (S)-(À)-a-terpineol, respectively. [16] The most likely candidate for proton abstraction in bCinS is Asn305, due to its close proximity to C7 and strong interaction with the water molecule during simulation. Asn is not a typical proton acceptor, but nevertheless, our high-level QM calculations suggest that proton transfer to the amide oxygen is favourable in this case.…”

De novo Transcriptome Characterization of Rhodomyrtus tomentosa Leaves and Identification of Genes Involved in α/β-Pinene and β-Caryophyllene Biosynthesis

“…GPP and FPP serve as substrates for terpene synthases (TPSs) for synthesizing mono- and sesqui-terpenes, respectively ( Yahyaa et al, 2015 ; Despinasse et al, 2017 ), during which the synthesis of monoterpenes is initiated by GPP dephosphorylation and ionization to geranyl carbocation, while the synthesis of sesquiterpene starts with FPP ionization to a farnesyl cation ( Degenhardt et al, 2009 ; Huang et al, 2010 ). This is then followed by a series of complex chemical mechanisms involving isomerizations, cyclizations, and rearrangements catalyzed by TPSs, which finally generate structurally diverse terpenoids ( Keszei et al, 2010 ; Koo et al, 2016 ; Piechulla et al, 2016 ) ( Figure 1 ).…”