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Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6‐Aminogalactose‐1‐Phosphate and Analogues
Author(s) -
Huang Kun,
Parmeggiani Fabio,
Pallister Edward,
Huang ChuenJiuan,
Liu FangFang,
Li Qian,
Birmingham William R.,
Both Peter,
Thomas Baptiste,
Liu Li,
Voglmeir Josef,
Flitsch Sabine L.
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700477
Subject(s) - galactokinase , biochemistry , chemistry , substrate (aquarium) , galactose , enzyme , phosphate , phosphorylation , escherichia coli , stereochemistry , biology , ecology , gene
Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate‐active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting‐group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii , which was produced in Escherichia coli and shown to catalyse 1‐phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1‐phosphorylation of a number of galactose analogues, including 3‐deoxy‐3‐fluorogalactose and 4‐deoxy‐4‐fluorogalactose, which were first reported to be substrates for wild‐type galactokinase. LgGalK and galactose oxidase variant M 1 were combined in a one‐pot, two‐step system to synthesise 6‐oxogalactose‐1‐phosphate and 6‐oxo‐2‐fluorogalactose‐1‐phosphate, which were subsequently used to produce a panel of 30 substituted 6‐aminogalactose‐1‐phosphate derivatives by chemical reductive amination in a one‐pot, three‐step chemoenzymatic process.