title: Unraveling the Leloir pathway of Bifidobacterium bifidum: significance of the uridylyltransferases.
abstract: The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.
authors.affiliation: Centre of Expertise-Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Ghent University, Ghent, Belgium.
authors.forename: Frederik
authors.initials: F
authors.lastname: De Bruyn
authors.forename: Joeri
authors.initials: J
authors.lastname: Beauprez
authors.forename: Jo
authors.initials: J
authors.lastname: Maertens
authors.forename: Wim
authors.initials: W
authors.lastname: Soetaert
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001644:Bifidobacterium / Q000201:enzymology* / Q000254:growth & development; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005695:Galactosephosphates / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008895:Milk, Human / Q000737:chemistry; D008969:Molecular Sequence Data; D005810:Multigene Family; D009844:Oligosaccharides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism*; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005695:Galactosephosphates; D009844:Oligosaccharides; C029973:galactose-1-phosphate; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D006599:UDPglucose-Hexose-1-Phosphate Uridylyltransferase