Abstract
N-Acetylhexosamine 1-kinase (NahK) catalyzes the direct addition of a phosphate from adenosine 5'-triphosphate (ATP) to the anomeric position of N-acetylhexosamine and shows similar activity towards N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc). Herein we report the cloning, characterization, and substrate specificity studies of two NahKs from Bifidobacterium infantis ATCC15697 and Bifidobacterium longum ATCC55813, respectively. A new capillary electrophoresis assay method has been developed for enzyme activity assays. Both enzymes have a good expression level in E. coli (180–185 mg/L culture) and can tolerate diverse modifications at C2 of GlcNAc and GalNAc. Various GlcNAc derivatives with C6, both C2 and C6, as well as both C2 and C3 modifications are tolerable substrates for the newly cloned NahKs. Quite interestingly, despite of their low activities toward glucose and galactose, the activities of both NahKs are much higher for mannose and some of its C2, C4, and C6 derivatives. These NahKs are excellent catalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
Highlights
N-Acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are important monosaccharides broadly distributed in Nature
We found that in addition to previously reported N-Acetylhexosamine 1-kinase (NahK) substrates, various GlcNAc derivatives including those with C2-azido, C6-azido, and 6-O-sulfate groups are tolerable substrates for the newly cloned NahKs
NahKs from Bifidobacterium infantis ATCC#15697 (NahK_ATCC15697) and Bifidobacterium longum ATCC#55813 (NahK_ATCC55813) were each cloned as a C-His6-tagged fusion protein in a pET22b(+) vector
Summary
N-Acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) are important monosaccharides broadly distributed in Nature. GlcNAc plays an important role in plant organogenesis and invertebrate embryogenesis [1] It is an essential component of protein N-glycans and some important polysaccharides including chitin (the second most abundant carbohydrate after cellulose) [2,3], bacterial cell wall [4], and some glycosaminoglycans such as hyaluronic acid, keratan sulfate, and heparan sulfate/heparin [5,6,7].
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