8h6u
Class I sesquiterpene synthase BCBOT2 (complex)
Structural highlights
FunctionBOT2_BOTFU Presilphiperfolan-8-beta-ol synthase; part of the gene cluster that mediates the biosynthesis of botrydial (PubMed:14651630, PubMed:19035644, PubMed:19476353). Botrydial is necessary for colonization of plant tissue by the T4 strain (PubMed:19035644). It is a strain-dependent virulence factor since highly aggressive strains like SAS56 or B05 still retain substantial virulence when botrydial synthesis is impaired, since they produce also botcinic acid (PubMed:15986930). The first step of botrydial biosynthesis is performed by the sesquiterpene synthase BOT2 which catalyzes the cyclization of farnesyl diphosphate (FPP) to presilphiperfolan-8-beta-ol (PSP) (PubMed:19035644, PubMed:19476353). The cytochrome P450 monooxygenase BOT4 then catalyzes the hydroxylation at C-4 to give a probotryane intermediate (PubMed:27529428, PubMed:28617493). Acetylation of the hydroxyl at C-4 is carried out by the acetyltransferase BOT5, followed by the combined action of the P450 monooxygenases BOT3 and BOT1, to yield finally the glycol, via the regio- and stereospecific hydroxylations at C-10 and C-15 of the probotryane intermediates, respectively (PubMed:15986930, PubMed:27529428). The cleavage of the C10-C15 bond of probotryane skeleton is an intriguing and chemically important reaction, which could be mediated by some of the monooxygenases or by a combination of them (PubMed:27529428). It is possible that either BOT3 or BOT1 would oxidize either the 10- or the 15-hydroxy group to the hydroperoxide derivative, which would then undergo heterolytic fragmentation to give the dialdehyde botrydial (PubMed:27529428). Finally, the dehydrogenase BOT7 might be involved in the conversion of botrydial to dihydrobotrydial (PubMed:27721016).[1] [2] [3] [4] [5] [6] [7] Publication Abstract from PubMedThe cyclization of farnesyl diphosphate (FPP) into highly strained polycyclic sesquiterpenes is challenging. We here determined the crystal structures of three sesquiterpene synthases (STSs, namely, BcBOT2, DbPROS, and CLM1) catalyzing the biosynthesis of the tricyclic sesquiterpenes presilphiperfolan-8beta-ol (1), Delta(6)-protoilludene (2), and longiborneol (3). All three STS structures contain a substrate mimic, the benzyltriethylammonium cation (BTAC), in their active sites, providing ideal templates for quantum mechanics/molecular mechanics (QM/MM) analyses toward their catalytic mechanisms. The QM/MM-based molecular dynamics (MD) simulations revealed the cascade reactions toward the enzyme products, and different key active site residues that play important roles in stabilizing reactive carbocation intermediates along the three pathways. Site-directed mutagenesis experiments confirmed the roles of these key residues and concomitantly resulted in 17 shunt products (4-20). Isotopic labeling experiments addressed the key hydride and methyl migrations toward the main and several shunt products. These combined methods provided deep insights into the catalytic mechanisms of the three STSs and demonstrated how the chemical space of STSs can rationally be expanded, which may facilitate applications in synthetic biology approaches toward pharmaceutical and perfumery agents. Structural Insights into Three Sesquiterpene Synthases for the Biosynthesis of Tricyclic Sesquiterpenes and Chemical Space Expansion by Structure-Based Mutagenesis.,Lou T, Li A, Xu H, Pan J, Xing B, Wu R, Dickschat JS, Yang D, Ma M J Am Chem Soc. 2023 Apr 5. doi: 10.1021/jacs.3c00278. PMID:37018048[8] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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