Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I

Abstract

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Gal␤1-3GlcNAc) and galacto-N-biose (GNB; Gal␤1-3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K d values of 0.087 and 0.010 M, respectively, and the binding process is enthalpydriven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.Bifidobacteria are Gram-positive anaerobes naturally present in the dominant colonic microbiota and have been considered to be beneficial for human health. As probiotic agents, Bifidobacteria can prevent or alleviate infectious diarrhea through their effects on the immune system and promote the host resistance to colonization by pathogens (1). Carbon source compounds, including oligofructose, inulin, and raffinose, are used as food additives (prebiotics) to selectively promote the growth of Bifidobacteria in the gut (2, 3). Bifidobacteria predominate the intestinal flora of breastfed infants (4, 5), whereas bottle-fed infants do not show rapid colonization of these organisms (6). It has been widely accepted that oligosaccharides other than lactose in human milk (human milk oligosaccharides, HMOs) 4 play a key role in the growth of Bifidobacteria in the gut (7,8). However, it remains unknown what structure, in HMOs, constitutes the bifidus factor responsible for increasing the bifidobacterial population. Human milk is reported to contain more than 100 kinds of oligosaccharides, the building blocks of which are the following three basic core disaccharides: lactose (Gal␤1-4Glc), lacto-N-biose I (LNB; Gal␤1-3GlcNAc), and N-acetyllactosamine (LacNAc; Gal␤1-4GlcNAc). These oligosaccharides are often modified by sialic acid and/or L-fucose (7). Lacto-N-tetraose (LNT; Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc), which is formed by a ␤1-3 linkage between LNB and lactose, and fucosylated derivative (Fuc␣1-2Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc) are the major components of HMOs (9 -11).Recently, we found that Bifidobacterium longum JCM1217 has a unique metabolic pathway specific for LNB and galacto-N-biose (GNB, Gal␤1-3GalNAc), and we presented the hypothesis that the LNB residue in HMOs is the bifidus factor in breastfed infants (12). B...

Characterization of a Novel β-l-Arabinofuranosidase in Bifidobacterium longum

“…Kitaoka et al (19,20) proposed a metabolic pathway for GNB from core-1 mucin-type O-glycans and Gal-␤1,3-GlcNAc (LNB) from human milk oligosaccharides based on the characterization of the genes encoded in the GNB/LNB operon (BLLJ_1620-BLLJ_1626) of B. longum. Fushinobu et al (21,22) characterized the GNB/LNB-binding protein (BLLJ_1626) of an ATP-binding cassette (ABC)-type sugar transport system in the GNB/LNB pathway. As shown in supplemental Fig.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…79) Afterwards, our structural and thermodynamic analyses revealed the detailed molecular mechanism of its disaccharide binding. 80) Isothermal titration calorimetry measurements indicated that GL-BP strongly binds GNB (K a ¼ 9:7 Â 10 7 M À1 ) and LNB (K a ¼ 1:2 Â 10 7 M À1 ), but does not bind LacNAc, lactose, galactose, GlcNAc, or GalNAc. The affinity for a major HMO tetrasaccharide, LNT, was 1,000-fold weaker than that for GNB (K a ¼ 9:1 Â 10 4 M À1 ).…”

Prebiotic Effect of Lacto-N-biose I on Bifidobacterial Growth

“…We have determined that bifidobacteria possess a gene cluster encoding the unique metabolic pathway for lacto-N-biose I (Gal1-3GlcNAc), in which the disaccharide is transported to the cytoplasm through a specific ABC-type transporter, 12,13) phosphorolyzed by galacto-N-biose (Gal1-3GalNAc)/lacto-N-biose I phosphorylase (renamed from lacto-N-biose phosphorylase after the discovery of the lacto-N-biose I specific enzyme), [14][15][16] and finally metabolized in the glycolytic and aminosugar metabolic pathways. 17) Although lacto-N-biose I is not present in free disaccharide form in human milk, it occurs as a building unit of type I oligosaccharides, containing lacto-N-tetraose (Gal1-3GlcNAc1-3Gal1-4Glc) as the core structure.…”