SummaryThe probiotic Lactobacillus casei catabolizes galacto-N-biose (GNB) and lacto-N-biose (LNB) by using a transport system and metabolic routes different from those of Bifidobacterium. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Inactivation of gnbC (EIIC) or ptsI (Enzyme I) of the phosphoenolpyruvate : sugar phosphotransferase system (PTS) prevented the growth on those three carbohydrates, indicating that they are transported and phosphorylated by the same PTS Gnb . Enzyme activities and growth analysis with knockout mutants showed that GnbG (phospho-β-galactosidase) hydrolyses both disaccharides. However, GnbF (N-acetylgalactosamine-6P deacetylase) and GnbE (galactosamine-6P isomerase/deaminase) are involved in GNB but not in LNB fermentation. The utilization of LNB depends on nagA (Nacetylglucosamine-6P deacetylase), showing that the N-acetylhexosamine moieties of GNB and LNB follow different catabolic routes. A lacAB mutant (galactose-6P isomerase) was impaired in GNB and LNB utilization, indicating that their galactose moiety is channelled through the tagatose-6P pathway. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR, which binds to a 26 bp DNA region containing inverted repeats exhibiting a 2T/2A conserved core. The data represent the first characterization of novel metabolic pathways for human milk oligosaccharides and glycoconjugate structures in Firmicutes.

Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds in carbohydrates and glycoconjugates, with various outcomes depending not only on the acceptor molecules they bind but also on the type of glycosidic bond they form (C−O, C−N, C−S, or C−C). Here we show that the glucosyltransferase UGT1 from the indigo plant Polygonum tinctorium catalyzes either N-, O-, or S-glycosylation with similar rates. We solve the structure of the enzyme in complex with its donor and acceptor substrates and elucidate the molecular basis of N-, O-, and S-specificities using experimental mutagenesis and QM/MM simulations, revealing distinct mechanisms for N-, O-, and S-glycosylation. We also show that the active site can be engineered to increase or favor one of the three glycosylation activities over another. These results will foster the design of more active and specific enzyme variants for production of glycosides.

Human milk oligosaccharides (HMOs) are considered to play a key role in establishing and maintaining the infant gut microbiota. Lacto-N-triose forms part of both type 1 and type 2 HMOs and also of the glycan moieties of glycoproteins. Upstream of the previously characterized gene cluster involved in lacto-N-biose and galacto-N-biose metabolism from Lactobacillus casei BL23, there are two genes, bnaG and manA, encoding a ␤-N-acetylglucosaminidase precursor and a mannose-6-phosphate isomerase, respectively. In this work, we show that L. casei is able to grow in the presence of lacto-N-triose as a carbon source. Inactivation of bnaG abolished the growth of L. casei on this oligosaccharide, demonstrating that BnaG is involved in its metabolism. Interestingly, whole cells of a bnaG mutant were totally devoid of ␤-N-acetylglucosaminidase activity, suggesting that BnaG is an extracellular wall-attached enzyme. In addition to hydrolyzing lacto-N-triose into N-acetylglucosamine and lactose, the purified BnaG enzyme also catalyzed the hydrolysis of 3=-N-acetylglucosaminyl-mannose and 3=-N-acetylgalactosaminyl-galactose. L. casei can be cultured in the presence of 3=-N-acetylglucosaminyl-mannose as a carbon source, but, curiously, the bnaG mutant strain was not impaired in its utilization. These results indicate that the assimilation of 3=-N-acetylglucosaminyl-mannose is independent of BnaG. Enzyme activity and growth analysis with a manA-knockout mutant showed that ManA is involved in the utilization of the mannose moiety of 3=-N-acetylglucosaminyl-mannose. Here we describe the physiological role of a ␤-N-acetylglucosaminidase in lactobacilli, and it supports the metabolic adaptation of L. casei to the N-acetylglucosaminide-rich gut niche. G lycans in human milk are present as free oligosaccharides or conjugated to proteins and lipids (1, 2), and they have been proposed to directly influence the composition of the infant gut microbiota (3, 4). Furthermore, the free human milk oligosaccharides (HMOs), the third largest solid component in milk, act as prebiotics to promote colonization by beneficial bacterial species (5, 6). HMOs contain a lactose moiety (Gal␤1-4Glc) at their reducing end, which is elongated by ␤1,3-linked lacto-N-biose units (Gal␤1-3GlcNAc) to give the type 1 HMOs, including lacto-Ntetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc), or by ␤1,3/6-linked N-acetyllactosamine units (Gal␤1-4GlcNAc) to give the type 2 HMOs, such as lacto-N-neotetraose (Gal␤1-4GlcNAc␤1-3Gal␤1-4Glc). Further elongation of these core structures is made by the addition of fucose and sialic acid residues (1). Both types of HMOs contain a lacto-N-triose unit (GlcNAc␤1-3Gal␤1-4Glc), highlighting the importance of this trisaccharide in the total pool of HMOs. In addition, lacto-N-triose and other N-acetylhexosaminyl-oligosaccharides also form part of the structure of glycans conjugated to proteins and lipids present in human milk. The carbohydrate moieties of these molecules also have a prebiotic role, and besides the monosaccharides described abov...