Identification of N-acetylhexosamine 1-kinase in the complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum

Abstract

We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.

FIG. 1.

Schematic structure of the gene cluster between the BL1638 and BL1644 genes.

FIG. 2.

Scheme of the Leloir pathway.

FIG. 3.

Lineweaver-Burk plot of the NahK reaction with GlcNAc at ATP concentrations of 0.2 mM (closed circles), 0.4 mM (open squares), 0.6 mM (closed squares), and 1.0 mM (triangles). The kinetic parameters in the equation v = V[GlcNAc][ATP]/(K_mATPK_iGlcNAc + K_mATP[GlcNAc] + K_mGlcNAc[ATP] + [GlcNAc][ATP]) were calculated to be as follows: V = 1.90 μmol/min·mg protein, K_mGlcNAc = 0.10 mM, K_mATP = 0.13 mM, and K_iGlcNAc = 0.40 mM. v, initial reaction rate at the concentrations of the substrates; V, maximum velocity.

FIG. 4.

Scheme of the LNB/GNB pathway in B. longum. Solid arrow, reaction catalyzed by an enzyme encoded in the operon; dashed arrow, reaction catalyzed by an enzyme not encoded in the operon.