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. 2008 Jul;74(13):3996-4004.
doi: 10.1128/AEM.00149-08. Epub 2008 May 9.

Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure

Jun Wada  1 , Takuro Ando, Masashi Kiyohara, Hisashi Ashida, Motomitsu Kitaoka, Masanori Yamaguchi, Hidehiko Kumagai, Takane Katayama, Kenji Yamamoto
Affiliations

Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure

Jun Wada et al. Appl Environ Microbiol. 2008 Jul.

Erratum in

  • Appl Environ Microbiol. 2009 Oct;75(19):6414

Abstract

Breast-fed infants often have intestinal microbiota dominated by bifidobacteria in contrast to formula-fed infants. We found that several bifidobacterial strains produce a lacto-N-biosidase that liberates lacto-N-biose I (Galbeta1,3GlcNAc; type 1 chain) from lacto-N-tetraose (Galbeta1,3GlcNAcbeta1,3Galbeta1,4Glc), which is a major component of human milk oligosaccharides, and subsequently isolated the gene from Bifidobacterium bifidum JCM1254. The gene, designated lnbB, was predicted to encode a protein of 1,112 amino acid residues containing a signal peptide and a membrane anchor at the N and C termini, respectively, and to possess the domain of glycoside hydrolase family 20, carbohydrate binding module 32, and bacterial immunoglobulin-like domain 2, in that order, from the N terminus. The recombinant enzyme showed substrate preference for the unmodified beta-linked lacto-N-biose I structure. Lacto-N-biosidase activity was found in several bifidobacterial strains, but not in the other enteric bacteria, such as clostridia, bacteroides, and lactobacilli, under the tested conditions. These results, together with our recent finding of a novel metabolic pathway specific for lacto-N-biose I in bifidobacterial cells, suggest that some of the bifidobacterial strains are highly adapted for utilizing human milk oligosaccharides with a type 1 chain.

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Figures

FIG. 1.
FIG. 1.
The GNB/LNB pathway and related enzymes. LNB (Galβ1,3GlcNAc) and GNB (Galβ1,3GalNAc) liberated from HMOs with a type 1 chain and mucin glycoproteins by LnbB and EngBF, respectively, might be captured by GL-BP, imported into the cell, and then phosphorolytically cleaved into Gal 1-P and the respective N-acetylhexosamines (GlcNAc and GalNAc). The former can undergo glycolysis via the Leloir-like pathway (GalE and GalT), and the latter can enter the aminosugar metabolic cycle after phosphorylation by NahK. Prior to the actions of LnbB and EngBF, α-1,2-fucosyl residues were removed by AfcA.
FIG. 2.
FIG. 2.
Liberation of lacto-N-biose I from lacto-N-tetraose upon incubation with B. bifidum JCM1254 cells. (A) TLC analysis of reaction products. Cells grown in GAM medium (1 ml) were harvested by centrifugation and incubated in the reaction mixture (1 ml) containing 0.9 mM lacto-N-tetraose and 10 mM potassium phosphate buffer (pH 7.0) at 37°C. Samples (5 μl) were withdrawn at the indicated times. Lacto-N-tetraose incubated with boiled cells was used as a control. Carbohydrates were visualized by heating the plate after dipping it in orcinol-H2SO4 reagent (20). The arrows indicate the positions of standard sugars. (B) ESI-MS analysis of the reaction product. The spot corresponding to LNB was extracted from the TLC plate, lyophilized, and then subjected to ESI-MS analysis. A molecular ion peak appeared at m/z 406.0, which is consistent with the calculated mass for a sodium adduct of LNB (406.1).
FIG. 3.
FIG. 3.
Primary structure of LnbB from B. bifidum JCM1254. (A) Schematic representation of the domain structure. The amino acid numbering starts at the probable initiation codon. The domain of GH20, CBM32, and bacterial Ig-like 2 domain are depicted as dark-gray, shaded, and light-gray boxes, respectively. The black bars at the N-terminal and C-terminal ends indicate a signal peptide and a membrane anchor, respectively. (B) Multiple alignment of GH20 domains of LnbB from B. bifidum JCM1254 and its homologues, created by TCOFFEE (44) and BoxShade 3.21. The numbering starts at the initiation codon of each protein. Identical residues and conserved substitutions are highlighted in black and dark gray, respectively. The general acid/base residues identified by the structural and biochemical analyses of N-acetylhexosaminidases are marked by asterisks, and the residues involved in the binding of O-3, O-4, and O-6 of N-acetylhexosamine and the stacking tryptophan at the −1 subsite are enclosed by boxes. The organisms and accession numbers are as follows; BB, LnbB from B. bifidum JCM1254; SS, LnbB from Streptomyces sp. strain 142 (U40488); SP, β-N-acetylhexosaminidase from S. plicatus (AF063001); HS, β-N-acetylhexosaminidase from H. sapiens (NM_000521); SG, β-N-acetylhexosaminidase from S. gordonii FSS2 (AY450645).
FIG. 4.
FIG. 4.
Hydrolysis of lacto-N-tetraose into LNB and lactose by the recombinant LnbB. Lacto-N-tetraose was incubated in the absence (A) and presence (B) of the purified enzyme, and the reaction mixtures were subjected to ESI-MS. The molecular ion peak at m/z 730.8 corresponds to a sodium adduct of lacto-N-tetraose (calculated, 730.2) (A), and the molecular ion peaks of 365.5 and 406.5 are sodium adducts of lactose (calculated, 365.1) and LNB (calculated, 406.1) (B), respectively.
FIG. 5.
FIG. 5.
Transglycosylation and condensation activities of the recombinant LnbB. (A) Transglycosylation to various 1-alkanols. Lacto-N-tetraose (donor) was incubated without (lane 2) and with (lanes 3 to 6) 1-alkanols in the presence of the enzyme. Lane 1, standard sugars (LNT, lacto-N-tetraose). The acceptors were as follows: lane 3, methanol; lane 4, ethanol; lane 5, 1-propanol; and lane 6, 1-butanol. (B) HPLC analyses of transglycosylation (a) and condensation (b) reactions of LnbB. pNP-β-LNB (donor) was incubated with lactose (acceptor) in the absence (top) and presence (bottom) of the enzyme (a), and LNB and lactose were incubated in the absence (top) and presence (bottom) of the enzyme (b). The elution was monitored by measuring the absorbance of the N-acetyl group at 214 nm.
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