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

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 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.

Engineering Orthogonal Methyltransferases to Create Alternative Bioalkylation Pathways.

S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) catalyse the methylation of a vast array of small metabolites and biomacromolecules. Recently, rare carboxymethylation pathways have been discovered, including carboxymethyltransferase enzymes that utilise a carboxy-SAM (cxSAM) cofactor generated from SAM by a cxSAM synthase (CmoA). We show how MT enzymes can utilise cxSAM to catalyse carboxymethylation of tetrahydroisoquinoline (THIQ) and catechol substrates. Site-directed mutagenesis was used to create orthogonal MTs possessing improved catalytic activity and selectivity for cxSAM, with subsequent coupling to CmoA resulting in more efficient and selective carboxymethylation. An enzymatic approach was also developed to generate a previously undescribed co-factor, carboxy-S-adenosyl-l-ethionine (cxSAE), thereby enabling the stereoselective transfer of a chiral 1-carboxyethyl group to the substrate.

Advance in Anti-tumor Mechanisms of Shikonin, Alkannin and their Derivatives.

Shikonin, alkannin and their derivatives, the main ingredient of Lithospermum erythrorhizon and Arnebia euchroma (Royle) Johnst native to Inner Mongolian and Northwest of China respectively, hold promising potentials for antitumor effects via multiple-target mechanisms. This review will emphasize the importance of their antitumor activity in apoptosis, necroptosis and immunogenic cell death, and expound the relationship of their antitumor activity and naphthoquinone scaffold that could generate ROS and alkylating agent. Meanwhile, the antitumor mechanisms of naturally-occurring shikonin, alkannin and their derivatives, which were divided into the direct interaction involved in alkylating agent, covalently binding the DNA and protein, as well as the indirect interaction mediated by ROS, nonspecifically influencing the mitochondria or multiple signal pathways, will be systematically summarized and discussed.