Broadening the Substrate Specificity of a Sugar Kinase to Span l - and d -Sugars for Coupled Activation Reactions

Sugar-1-phosphates are key intermediates in the synthesis of nucleotide sugars, which serve as essential building blocks for glycans from prokaryotes to eukaryotes. Compared to mammalian glycomes, those of microbes are enriched in deoxy l-sugars, many of which are activated as nucleotide β-l-sugars in the cell. The in vitro and cellular construction of many biologically relevant microbial polysaccharides, natural product glycoconjugates, surface glycoproteins, and antigens relies on nucleotide β-l-sugars. Yet, there remain few efficient chemical or biochemical routes toward the activated forms of these sugars or their sugar-1-phosphate starting materials. Here, we perform rational and unbiased engineering of the sugar kinase domain of the microbial l-fucose salvage enzyme FKP (Fucokinase/GDP-fucose pyrophosphorylase) and expand its substrate scope to span noncanonical l- and d-monosaccharides. We show that point mutations outside of the active site enable efficient phosphorylation of the prokaryote-specific sugar 6-deoxy-l-talose, and an active site mutation facilitates the phosphorylation of d-glucose. Coupling of FKP and its mutants with nucleotidyltransferases facilitates the production of diverse nucleotide sugars in one pot, highlighting facile in vitro methods to produce necessary starting materials that can be used in the enzymatic production of microbial and eukaryotic glycans.