Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs

Abstract

Biocatalytic alkylations are important reactions to obtain chemo-, regio-and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

A bicyclic S-adenosylmethionine regeneration system applicable with different nucleosides or nucleotides as cofactor building blocks

“…35 Based on the previously stated limitations of the cyclic system, the starting point of this work was to improve the SAM regeneration cycle by (i) avoiding the formation of adenine and/or (ii) removing L-homocysteine from the system. As a first model, the catechol O-MT from Rattus norvegicus (RnCOMT) was used for the conversion of 3, 4-dihydroxybenzoic acid (12) to vanillic acid (13) and isovanillic acid (14), with 13 being the main regioisomer formed (Fig. 2A).…”

“…6,7 Commonly used chemical methylation agents such as methyl iodide or dimethyl sulfate are often carcinogenic, mutagenic, and harmful to the environment. The biocatalytic application of MTs, also using SAM analogues, [8][9][10][11][12] are promising starting points for sustainable methods. 13,14 While SAM analogues allowing to transfer larger alkyl chains have been widely employed for product diversification, [15][16][17][18][19][20][21][22][23][24][25] the variation of the nucleobase has become a new focus, especially regarding the long-term goal to make bioorthogonal systems available.…”

From Natural Methylation to Versatile Alkylations Using Halide Methyltransferases

“…These HMTs, combined with alkyl‐promiscuous MTs, produced the regioselectively alkylated products 4′‐ O ‐ethylluteolin, 4‐allyloxy‐3‐hydroxybenzaldehyde, 1‐ethyl‐5‐cyclopropylpyrazole, and 1–propyl‐5‐cyclopropylpyrazole (Scheme 3c). In our work, more than 40 regeneration cycles were achieved for the preparative scale alkylation of flavonoid and phenolic compounds, using 100 μM of SAH [21] . Importantly, these cycle numbers may well be underestimated, as additional SAH added to the reactions may not have been necessary.…”

Recent trends in biocatalysis

“…This can be sufficient if single site-saturation or small combinatorial libraries have to be screened. 29,30 While these methods are still applicable to larger random and combinatorial libraries, they are not capable of screening a significant fraction of the library diversity. 31 One solution to this problem is to make the wells smaller, going through 1536-well plates to microcapillary arrays.…”

“…[209][210][211] A more advanced way is to explore promiscuous or engineered HMTs for the production of SAM analogues and to achieve flavonoid bioalkylation on the basis of the MT-HMT cofactor regeneration system. 30,212 This artificial cofactor regeneration pathway provides a novel inspiration and solution for solving the problem of low efficiency of SAM regeneration in biosynthesis.…”