A chemoenzymatic platform for the synthesis ofS‐adenosyl‐L‐methionine (SAM) analogues compatible with downstream SAM‐utilizing enzymes is reported. Forty‐four non‐native S/Se‐alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MAT II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non‐native SAM analogues. As a proof of concept for the feasibility of natural product “alkylrandomization”, a small set of differentially‐alkylated indolocarbazole analogues was generated by using a coupled hMAT2–RebM system (RebM is the sugar C4′‐O‐methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.

Covering: up to 2012 The glycosylation of microbial natural products often dramatically influences the biological and/or pharmacological activities of the parental metabolite. Over the past decade, crystal structures of several enzymes involved in the biosynthesis and attachment of novel sugars found appended to natural products have emerged. In many cases, these studies have paved the way to a better understanding of the corresponding enzyme mechanism of action and have served as a starting point for engineering variant enzymes to facilitate to production of differentially-glycosylated natural products. This review specifically summarizes the structural studies of bacterial enzymes involved in biosynthesis of novel sugar nucleotides.

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k _cat /K _m of >400-fold and >15-fold for formation of NDP–glucoses and UDP–sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP–sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.