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.

Fig 1

Schematic representation of the GNB/LNB gene cluster in B. longum NCC2705 (gene BL1638 to BL1644) and B. bifidum PRL2010 (gene BBPR_1050 to BBPR_1058). Two sugar kinases interrupt the GNB/LNB pathway of B. bifidum, and it is not organized as in the lnpABCD operon (BL1641 to BL1644) of B. longum.

Fig 2

Overview of the Leloir pathway (A) and GNB/LNB pathway (B) in Bifidobacterium bifidum. LNBP, NahK, GalT2, and GalE2 are encoded in the same gene cluster (4). In contrast, the classic Leloir pathway (A) is encoded by scattered genes, and evidence is lacking for the existence of metabolic crossover between both pathways. Gene locus tags of the corresponding enzymes are listed in Table 1.

Fig 3

16S rRNA-based phylogenetic tree with gene cluster organization and prevalence of class I enzymes (GalT1 type) and class II enzymes (GalT2 type) in three representative bacterial phyla. The bacterial strain codes and bootstrapping values are included. Orthologues that match the galT1 or galT2 genes of B. bifidum are aligned vertically. GalT2-type enzymes are present only in Firmicutes and Actinobacteria and show a high degree of rearrangement with the neighboring genes.

Fig 4

Proposed routes for the breakdown of galactose, lacto-N-biose I (LNB), and galacto-N-biose (GNB) in B. bifidum. All 3 substrates yield gal1P, which is metabolized solely by the Leloir pathway (left). Depending on the presence of LNB or GNB, N-acetylglucosamine (glcNAc) or N-acetylgalactosamine (galNAc), respectively, is formed through the action of LNBP. Utilization of glcNAc requires only N-acetylhexosamine kinase (NahK) activity to generate glcNAc1P. However, GNB-rich substrates such as mucin need the full set of enzymes encoded by the GNB/LNB gene cluster (right). galNAc is phosphorylated to galNAc1P by NahK, which is subsequently converted by GalT2 and GalE2 via a Leloir-like pathway to glcNAc1P, which can then enter the bifid shunt via fructose 6-phosphate (F6P) by consequent action of a glucosamine mutase, a deacetylase, and a deaminase. glcN6P, glucosamine 6-phosphate.