Effective Synthesis of Guanosine 5′‐Diphospho‐β‐ l ‐galactose Using Bacterial l ‐Fucokinase/Guanosine 5′‐Diphosphate‐ l ‐fucose Pyrophosphorylase

The nucleotide sugar guanosine 5′‐diphospho‐β‐ l ‐galactose (GDP‐ l ‐Gal) is known as a key intermediate of the l ‐ascorbic acid biosynthesis pathway of plants and algae. In addition, GDP‐ l ‐Gal serves as a donor substrate of l ‐galactosyltransferase, which transfers l ‐Gal on the non‐reducing ends of glycoconjugates. To synthesize varieties of l ‐Gal‐containing glycoconjugates and explore the novel l ‐galactosyltransferases, GDP‐ l ‐Gal needs to be prepared since it is not commercially available. In plants, GDP‐ d ‐mannose‐3′,5′‐epimerase (GME) converts GDP‐α‐ d ‐mannose (GDP‐ d ‐Man) to GDP‐ l ‐Gal and GDP‐ l ‐gulose. GDP‐ l ‐Gal has been previously prepared using GME and GDP‐ d ‐Man, for which the equilibrium ratio was biased to GDP‐ d ‐Man. In this study, the efficient GDP‐ l ‐Gal production from l ‐Gal was established using bacterial l ‐fucokinase/GDP‐ l ‐fucose pyrophosphorylase (FKP), l ‐fucose ( l ‐Fuc), adenosine 5′‐triphosphate (ATP) and guanosine 5′‐triphosphate (GTP). A high synthesis yield was obtained after screening of the optimum FKP reaction conditions in a 96‐well format and analysis by multiplexed capillary electrophoresis (MP‐CE). Conversion of l ‐Gal substrate yielded 97% GDP‐ l ‐Gal using purified recombinant FKP expressed in E. coli . Moreover, GDP‐ l ‐Gal was successfully purified with 92% (34.5 mg) overall yield from the FKP reaction mixture. GDP‐ l ‐Gal is now readily available for studies on l ‐galactosyltransferases and l ‐fucosyltransferases.