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Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3
Author(s) -
Li SiPeng,
Hsiao WeiChen,
Yu ChingChing,
Chien WeiTing,
Lin HongJyune,
Huang LiDe,
Lin ChienHung,
Wu WanLing,
Wu ShihHsiung,
Lin ChunCheng
Publication year - 2014
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/adsc.201400066
Subject(s) - chemistry , biochemistry , thermostability , glycoconjugate , antigen , enzyme , galactokinase , uridine diphosphate glucose , glycosyl , galactosyltransferase , galactose , uridine diphosphate , stereochemistry , biology , escherichia coli , gene , genetics
Herein, we report the first characterization of a novel galactokinase from Meiothermus taiwanensis sp. nov. WR‐220 (MtGalK), which is overexpressed in E. coli and exhibits a k cat / K m value of 168.47 mM −1 s −1 toward Gal at 75 °C. The thermophilic MtGalK shows specific substrate recognition toward the Gal configuration with limited tolerance for modifications at the C‐2 position to form products such as GalN‐1‐P, GalN 3 ‐1‐P, and GalNAc‐1‐P. Due to its unique thermostability toward elevated reaction temperatures, MtGalK served as an ideal biocatalyst in the synthesis of useful sugar‐1‐phosphates and was further combined with glucose‐1‐phosphate thymidylyltransferase (RmlA) and α‐1,4‐galactosyltransferase (LgtC) to achieve the efficient preparative‐scale synthesis of globotriose analogs (Gb3) using a one‐pot three‐enzyme system. In combination with a chemical synthetic strategy, a carbohydrate antigen from breast cancer stem cells, stage specific embryonic antigen‐3 (SSEA‐3) pentasaccharide, was synthesized from Gb3 in ten steps with a 23% overall yield. This flexible chemoenzymatic strategy for the synthesis of SSEA‐3 also allowed us to further expand the inventory of valuable natural and non‐natural glycans.