Functional Characterization of a Regiospecific Sugar-O-Methyltransferase from Nocardia

Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2'-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. IMPORTANCE ThnM1 is a putative sugar-O-methyltransferase produced by the Nocardia sp. strain CS682 and is encoded by a gene belonging to the biosynthetic gene cluster (BGC) of 1-(α-l-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene. We demonstrated that ThnM1 is a promiscuous enzyme with regiospecific activity at the 2'-OH of rhamnose. As regiospecific methylation of sugars by chemical synthesis is a challenging step, ThnM1 may fill the gap in the potential diversification of natural products by methylating the rhamnose moiety attached to them.