Reaction mechanism of the farnesyl pyrophosphate C-methyltransferase towards the biosynthesis of pre-sodorifen pyrophosphate by Serratia plymuthica 4Rx13

Abstract

Classical terpenoid biosynthesis involves the cyclization of the linear prenyl pyrophosphate precursors geranyl-, farnesyl-, or geranylgeranyl pyrophosphate (GPP, FPP, GGPP) and their isomers, to produce a huge number of natural compounds. Recently, it was shown for the first time that the biosynthesis of the unique homo-sesquiterpene sodorifen by Serratia plymuthica 4Rx13 involves a methylated and cyclized intermediate as the substrate of the sodorifen synthase. To further support the proposed biosynthetic pathway, we now identified the cyclic prenyl pyrophosphate intermediate pre-sodorifen pyrophosphate (PSPP). Its absolute configuration (6R,7S,9S) was determined by comparison of calculated and experimental CD-spectra of its hydrolysis product and matches with those predicted by semi-empirical quantum calculations of the reaction mechanism. In silico modeling of the reaction mechanism of the FPP C-methyltransferase (FPPMT) revealed a SN2 mechanism for the methyl transfer followed by a cyclization cascade. The cyclization of FPP to PSPP is guided by a catalytic dyad of H191 and Y39 and involves an unprecedented cyclopropyl intermediate. W46, W306, F56, and L239 form the hydrophobic binding pocket and E42 and H45 complex a magnesium cation that interacts with the diphosphate moiety of FPP. Six additional amino acids turned out to be essential for product formation and the importance of these amino acids was subsequently confirmed by site-directed mutagenesis. Our results reveal the reaction mechanism involved in methyltransferase-catalyzed cyclization and demonstrate that this coupling of C-methylation and cyclization of FPP by the FPPMT represents an alternative route of terpene biosynthesis that could increase the terpenoid diversity and structural space.

Non‐canonical Biosynthesis of the Brexane‐Type Bishomosesquiterpene Chlororaphen through Two Consecutive Methylation Steps in Pseudomonas chlororaphis O6 and Variovorax boronicumulans PHE5‐4

“…The proposed reaction mechanism (Scheme 1), initiated by SAM-dependent C-methylation of FPP (3 a) at C10, followed by cyclization and ring contraction, [5,6] As expected for terpene cyclases resulting in different products, the amino acid sequence identities between Pc-ChloS and Sp-SodS are limited to 17.66 % (Figure S12). The second putative TPS within the P. chlororaphis O6 gene cluster (PchlO6_6044) is more similar to sodorifen (Sp-SodS; 41.96 % identity; Figure S13), but did not produce any cyclization product with FPP (3 a; or GPP) or α-PSPP (2 a) upon coupled enzyme assays with Sp-FPP-MT, or γ-PSPP (4 a) and α-PCPP (5 a) when co-expressed with both C-methyltransferases from P. chlororaphis O6 (Figure S3), and is considered to be nonfunctional.…”

Enzymatic C3-Methylation of Indoles Using Methyltransferase PsmD─Crystal Structure, Catalytic Mechanism, and Preparative Applications

“…Specifically, we suggest that Y128 is deprotonated through a Glu-His-Tyr proton shuttle system. Variations of this system were reported in other MTases, with a histidine residue often present as a general base, in some cases accompanied by activating acidic residues. ,− Similarly, catalytic dyads containing adjacent histidine and tyrosine residues have been reported in several MTases that display an acid–base mechanism of activation prior to methylation. − In PsmD, the negatively charged Y128 is suggested to activate the substrate by engaging the proton attached to the indole nitrogen, increasing the electron density on the indole ring and triggering the nucleophilic attack of C3 on the methyl group of SAM (Scheme a). Substrate activation could be achieved via hydrogen bonding or deprotonation by the Y128 phenolate anion.…”

Biocatalytic One-Carbon Transfer – A Review

“…Lemfack, Brandt, and co-workers described in 2021 the biosynthesis of sodorifen, in which methylation catalyzed by farnesyl pyrophosphate MTs is essential for the subsequent cyclization reaction. 148 Geranyl pyrophosphate MTs was studied in the biosynthesis of 2-methylisoborneol by Dickschat and co-workers, 149 and isoprenyl phosphate MTs was used to expand the isoprenoid building block repertoire by Buchhaupt and coworkers. 150 The potential of MTs in synthetic applications and drug design is evident; however, in vitro applications are limited by their requirement for SAM as a stoichiometric methyl donor.…”