Synthesis of nucleotide-activated oligosaccharides by beta-galactosidase from Bacillus circulans.

Three decades ago nucleotide-activated oligosaccharides were isolated from milk of mammals, including man, and characterized as elongated UDP-N-acetyl-α-D-glucosamine (UDP-GlcNAc) derivatives (Jourdian et al., 1961; Kobata, 1962, 1963, 1966; Kobata and Ziro, 1965). Goat colostrum turned out to contain Neu5Ac-Gal(16)GlcNAc-UDP and Neu5Ac-Gal(1-4)GlcNAc-UDP (Jourdian et al., 1961). In human milk and colostrum Gal(β14)GlcNAc-UDP and Fuc(1 – 2 or 4)Gal(β1-4)GlcNAc-UDP were found (Kobata, 1963, 1966). Nucleotide-activated disaccharides were also isolated from hen oviduct and identified as Gal(β1-P-6)GlcNAc(α1-UDP) and Fuc(14)GlcNAc-UDP (Suzuki, 1962; Nakanishi et al., 1967). Although these glycoconjugates are known to be present in higher animals, their biosynthesis and biological function have not yet been elucidated. In archaebacteria UDPand GDP-activated oligosaccharides were identified as precursors in the biosynthesis of cell wall components such as pseudomurein (e. g. GalNAc(β1-3)GlcNAc-UDP) and the S-layer (e. g. saccharide-GlcNAc-UDP, saccharide-Gal-UDP, saccharideManNAc-GDP; Hartmann and Konig, 1989; Konig et al., 1989, 1994; Hartmannet al., 1993). However, the biosynthetic route is still not known and the enzymes involved have not yet been isolated and characterized. In this context an access to nucleotide-activated oligosaccharides by chemical and/or enzymatic methods could help to elucidate the biosynthesis and biological function of these naturally occurring glycoconjugates. In contrast to earlier findings (Jourdian and Distler, 1973), we have recently demonstrated that UDP-GlcNAc is also an in vitro acceptor substrate for β-1,4-galactosyltransferase (β4GalT1, EC 2.4.1.38) from human and bovine milk, resulting in the formation of Gal(β1-4)GlcNAc(α1UDP) (UDP-LacNAc; Elling et al. , 1999). This result implies that β4GalT1 may be responsible for the biosynthesis of UDP-LacNAc found in human milk. Jourdian and Distler (1973) reported the enzymatic in vitro synthesis of the UDP-disaccharides Gal(β1-3, 4 or 6)GlcNAc-UDP using the transglycosylation activity of βgalactosidase from bovine testes with UDP-GlcNAc as acceptor substrate. Since these products could be further sialylated by incubation with a goat colostrum sialyltransferase and CMP-N-acetylneuraminic acid, it was proposed that this might be the pathway for the enzymatic synthesis of UDP-disaccharides and UDP-trisaccharides (Jourdian and Distler, 1973). In order to investigate improvements in the regioselectivity, we have started to test several glycosidases with Biol. Chem., Vol. 382, pp.299 – 311, February 2001 · Copyright © by Walter de Gruyter · Berlin · New York