Rational reprogramming of the sesquiterpene synthase BcBOT2 yields new terpenes with presilphiperfolane skeleton

Abstract

Computer-aided rational design of the substrate binding pocket of sesquiterpene synthases BcBOT2 from Botrytis cinerea yielded FPP cyclization products with presilphiperfolane backbone other than the naturally formed sesquiterpene presilphiperfolan-8β-ol. Particularly,...

Chemoenzymatic Synthesis of a New Germacrene Derivative Named Germacrene F

“…The reaction intermediates resulting from the 1!10 transformation yield a tertiary carbocation, a structural motif exhibiting superior stability compared to the secondary carbocation arising from the 1!11 conversion. Notably, in agreement with the investigations by Christianson [25] carbocation intermediates derived from FPP and its derivatives [26] experience stabilization through π-system interactions mainly through phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) amino acid residues. Our molecular docking analysis (Figure 2) revealed that the reaction intermediate stemming from the cyclization event 1!10 predominantly engages in arene-H interactions with the neighbouring Phe81 residue, with occasional occurrences involving Trp303.…”

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Lifecycle of Botrytis cinerea

“…The polyketides and sesquiterpenes isolated from the culture media of the ∆Bcstc3 and OvBcstc3 strains were characterized by extensive spectroscopic and spectrometric mass analyses (Supplementary Figure S6). Their spectroscopic and physics constants were identical to those obtained from authentic samples isolated via OSMAC methodology from several previously reported B. cinerea strains [30][31][32][47][48][49].…”

“…The aspartate-rich motif was identified at position 177 for the first aspartate residue and the NSE/DTE motif at position 307 for the asparagine residue (Supplementary Figures S1 and S3). Two other motifs involved in catalysis were also identified in the BcStc3 sequence: the conserved effector triad (R264-D267-I268 residues) that also coordinates the PPi, aiding in substrate recognition and is associated with the initiation of the cyclization reaction [9,10,40], and the conserved RY pair (R334-Y335 residues) (Supplementary Figures S1 and S3) that is involved in the PPi recognition through hydrogen bonds and the formation of the caryophyllenyl cation [40,49]. Two of the three residues involved in the selectivity of BcStc1/BcBot2 (W94, F99 and N325 residues) [47] were also identified in the sequence of BcStc3 (F174 and N392 residues) (Supplementary Figure S1).…”

Unravelling the Function of the Sesquiterpene Cyclase STC3 in the Life Cycle of <em>Botrytis cinerea</em>

“…The aspartate-rich motif was identified at position 177, for the first aspartate residue, and at residue 307, for the N of the NSE/DTE motif (Figure 1). Two other motifs involved in catalysis were also identified in the BcStc3 sequence: the conserved effector triad (R264-D267-I268 residues) that also coordinate the ppi, aiding in substrate recognition and is associated with the initiation of the cyclization reaction [47,56,57], and the conserved RY pair (R334-Y335 residues) (Figure 1) that is involved in the PPi recognition through hydrogen bonds [47,59]. The arginine residue in the RY pair of BcStc1/BcBot2 (R373) plays a significant role after the formation of the caryophyllenyl cation [59].…”

“…Two other motifs involved in catalysis were also identified in the BcStc3 sequence: the conserved effector triad (R264-D267-I268 residues) that also coordinate the ppi, aiding in substrate recognition and is associated with the initiation of the cyclization reaction [47,56,57], and the conserved RY pair (R334-Y335 residues) (Figure 1) that is involved in the PPi recognition through hydrogen bonds [47,59]. The arginine residue in the RY pair of BcStc1/BcBot2 (R373) plays a significant role after the formation of the caryophyllenyl cation [59]. Two of the three residues involved in the selectivity of BcStc1/BcBot2 (W94, F99 and N325 residues) [47] were identified in the sequence of BcStc3 (F174 and N392 residues, Figure 1), probably also related to the selectivity of the enzyme.…”