Highly efficient chemoenzymatic synthesis of β1–4-linked galactosides with promiscuous bacterial β1–4-galactosyltransferases

Abstract

An efficient one-pot multienzyme approach has been developed for the synthesis of structural diverse LacNAc, lactose, and their derivatives including those containing negatively charged 6-O-sulfated GlcNAc and C2-substituted GlcNAc or Glc analogs. Two bacterial β1–4-galactosyltransferases, NmLgtB and Hp1–4GalT, exhibits promiscuous and complementary acceptor substrate specificity. The application of these enzymes in the one-pot multienzyme system allows the access to complex disaccharides with diverse structural modifications from monosaccharide derivatives and inexpensive Glc-1-P without using expensive sugar nucleotide. Bacterial carbohydrate-biosynthetic enzymes have been proven as efficient tools in novel synthesis of diverse and complex carbohydrates.

Biotechnological advances in UDP-sugar based glycosylation of small molecules

“…Andreana et al, 2002) for the formation of novel monosaccharides,643 but commonly available sugars may also be chemically modified 644(Cai et al, 2009a,b;Lau et al, 2010). Activation of these sugars 645 is achieved by coupling with promiscuous (anomeric) kinases and 646 uridylyltransferases to yield the corresponding UDP-sugars(Fig.…”

Sialoside Arrays: New Synthetic Strategies and Applications

“…In another piece of work reported by Chen and co-workers, two bacterial β-1,4-galactosytransferases, NmLgtB and Hp1-4GalT, showed promiscuous and complementary specificity for GlcNAc monosaccharide substrates with 6-O-sulfation or N-sulfation [84]. Using these enzymes in the one-pot multienzyme synthesis efficiently generated an array of LacNAc and lactose derivatives [84].…”

“…Using these enzymes in the one-pot multienzyme synthesis efficiently generated an array of LacNAc and lactose derivatives [84]. Moreover, chemoenzymatic synthesis of sulfur-linked saccharides was also investigated [85].…”

Glucose 1‐Phosphate Thymidylyltransferase in the Synthesis of Uridine 5′‐Diphosphate Galactose and its Application in the Synthesis ofN‐Acetyllactosamine

“…Although the expression of mammalian GalTs by E. coli has been reported, these attempts have mainly produced insoluble forms of the enzymes that were difficult to reactivate in large amounts; however, in comparison to mammalian b-1,4-GalTs, bacterial GalTs have the advantage of easy production in E. coli and better tolerance to substrate modification. [24] Previous studies have shown that b-1,4-GalT from Neisseria meningitides loses its activity when a histidine tag is fused to either its N-or C-terminus. [25] To facilitate the purification step of the GalT in this study, the b-1,4-GalT gene (lgtB) was cloned and then overexpressed in E. coli by an intein-mediated protein purification system.…”