Lacto-N-biosidase encoded by a novel gene of Bifidobacterium longum subspecies longum shows unique substrate specificity and requires a designated chaperone for its active expression

Abstract

Infant gut-associated bifidobacteria possess species-specific enzymatic sets to assimilate human milk oligosaccharides, and lacto-N-biosidase (LNBase) is a key enzyme that degrades lacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc), the main component of human milk oligosaccharides, to lacto-N-biose I (Galβ1-3GlcNAc) and lactose. We have previously identified LNBase activity in Bifidobacterium bifidum and some strains of Bifidobacterium longum subsp. longum (B. longum). Subsequently, we isolated a glycoside hydrolase family 20 (GH20) LNBase from B. bifidum; however, the genome of the LNBase(+) strain of B. longum contains no GH20 LNBase homolog. Here, we reveal that locus tags BLLJ_1505 and BLLJ_1506 constitute LNBase from B. longum JCM1217. The gene products, designated LnbX and LnbY, respectively, showed no sequence similarity to previously characterized proteins. The purified enzyme, which consisted of LnbX only, hydrolyzed via a retaining mechanism the GlcNAcβ1-3Gal linkage in lacto-N-tetraose, lacto-N-fucopentaose I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc), and sialyllacto-N-tetraose a (Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Gal); the latter two are not hydrolyzed by GH20 LNBase. Among the chromogenic substrates examined, the enzyme acted on p-nitrophenyl (pNP)-β-lacto-N-bioside I (Galβ1-3GlcNAcβ-pNP) and GalNAcβ1-3GlcNAcβ-pNP. GalNAcβ1-3GlcNAcβ linkage has been found in O-mannosyl glycans of α-dystroglycan. Therefore, the enzyme may serve as a new tool for examining glycan structures. In vitro refolding experiments revealed that LnbY and metal ions (Ca(2+) and Mg(2+)) are required for proper folding of LnbX. The LnbX and LnbY homologs have been found only in B. bifidum, B. longum, and a few gut microbes, suggesting that the proteins have evolved in specialized niches.

Keywords: Bacterial Metabolism; Carbohydrate Metabolism; Glycobiology; Glycoside Hydrolases; Microbiology; Oligosaccharide.

FIGURE 1.

Genetic organization of the lnbXY locus in the genome of B. longum subsp. longum JCM1217. Shown is the shortest region contained within the LNBase⁺-E. coli transformants. The locus tag, the annotated functions, and the Pram motifs are indicated. The numbering is based on GenBank^TM accession number NC_015067.

FIGURE 2.

Complementation analysis of lnbX-deficient B. longum subsp. longum. The strain carrying a disrupted lnbX gene was transformed with a plasmid carrying lnbX, lnbY, or lnbXY. Note that lnbY was expressed under the control of the lnbX promoter. The cells were grown in GAM medium overnight, harvested, and then suspended in basal medium (17) containing 5 mm LNT. The suspensions were incubated anaerobically for 1 h, and the supernatants were analyzed by thin layer chromatography using a solvent system of 1-butanol/acetic acid/water (2:1:1 by volume). The sugars were visualized by diphenylamine-phosphoric acid (45). Lane 1, JCM1217; lane 2, 105-A; lane 3, 105-A lnbX::Sp^R; lane 4, pTK2064 (empty vector)/lnbX::Sp^R; lane 5, pTK2064-lnbX⁺/lnbX::Sp^R; lane 6, pTK2064-lnbY⁺(lnbXp)/lnbX::Sp^R; lane 7, pTK2064-lnbX⁺Y⁺/lnbX::Sp^R. Standards used were LNT, Lac, and LNB.

FIGURE 3.

Purification of non-tagged, recombinant LnbX and LnbY proteins. a, the results of SDS-polyacrylamide gel electrophoresis. LNBase (LnbX) was purified by monitoring LNB-β-pNP-hydrolyzing activity. The N-terminal five amino acid residues of LnbX are MQSAT, determined by Edman degradation. The purified proteins were examined for their metal content (see “Results”). b, the native molecular weights of LnbX and LnbY were determined by size exclusion chromatography using Superdex 200 10/300 GL (for LnbX) and Superdex 75 10/300 GL (for LnbY) columns. Thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), ribonuclease A (13.7 kDa), and aprotinin (6.5 kDa) were used as standards.

FIGURE 4.

Substrate specificity of LNBases from B. longum subsp. longum (LnbX) and from B. bifidum (LnbB, a GH20 enzyme). The reactions were carried out in 50 mm MES (pH 5.4) (for LnbX) and citrate-phosphate (pH 4.5) (for LnbB) buffers containing 5 mm substrates in the presence and absence of the enzyme (6 milliunits toward LNB-β-pNP). The reaction products were analyzed by thin layer chromatography using a solvent system of 1-butanol/acetic acid/water (2:1:1 by volume). The sugars were visualized by diphenylamine-phosphoric acid (45). The substrates used were LNT (a), LNnT (b), lacto-N-triose II (LNTri II) (c), LNH (d), LNFP I (e), LNFP II (f), LST a (g), and LST b (h). *1 and *2 indicate 3′-sialyl-lacto-N-biose I (hydrolysis product, see Fig. 5c) and LNT (contaminant of LST b), respectively. Standards (Std.) used are lactose and each substrate (far left lane) and lacto-N-biose I (near left lane). See also Table 3.

FIGURE 5.

Electrospray ionization-MS analysis of the reaction products catalyzed by LnbX. The reactions were carried out in 50 mm MES (pH 5.4) buffer containing 5 mm LNT (a), LNFP I (b), and LST a (c) in the presence of the enzyme. When LNT and LNFP I were used as substrates, the reaction products were deionized by Amberlite MB-3 (Millipore) and lyophilized prior to the analysis. In LST a hydrolysis, the reaction products were first separated by thin layer chromatography, and the spot corresponding to a trisaccharide was extracted and purified by Sep-Pak C18 Plus (Waters, MA). Control experiments were performed without the enzyme (data not shown).

FIGURE 6.

Stereochemical course of the hydrolysis catalyzed by LnbX. The reaction mixture containing 50 mm MES buffer (pH 5.4), 2 mm LNB-β-pNP, and the enzyme (7 milliunits) was incubated at 30 °C and sampled at the indicated times to be analyzed by an HPLC system equipped with TSKgel Amide-80 (4.6 × 250 mm). Elution was carried out at room temperature using acetonitrile/water (65:35) flowing at 1 ml/min and was monitored by measuring the absorbance at 214 nm.

FIGURE 7.

Requirement of LnbY for proper folding of LnbX. a, the results of SDS-polyacrylamide gel electrophoresis of the soluble fraction of the cell-free extracts of E. coli cells expressing either LnbX(His) alone or LnbX(His) and LnbY. b, the cell-free extracts were separated by size exclusion chromatography (Superdex 200 10/300 GL). Each fraction (0.5 ml) of the elution volume (7.5–13 ml) was subjected to SDS-polyacrylamide gel electrophoresis, followed by Western blot analysis using anti-His tag antibodies linked to horseradish peroxidase. The fractions were also assayed for protein (mg/ml) and enzyme activity (units/mg). The peak fractions of the marker proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa) and the void fraction are indicated. c, in vitro refolding of denatured LnbX (1 μm) was carried out in the presence and absence of LnbY (1 μm) and the metal ions (0.1 mm CaCl₂ and MgCl₂). The refolding mixture comprising denatured LnbX alone (open circles); denatured LnbX and metal ions (filled circles); denatured LnbX and non-denatured LnbY (open squares); or denatured LnbX, non-denatured LnbY, and metal ions (filled squares) was dialyzed against 50 mm HEPES buffer containing or not containing 0.1 mm metal ions. The samples were removed from the dialysis cassettes at the indicated times and assayed for activity content. d, the refolding was carried out in varying concentrations of LnbY in the presence of 0.1 mm metal ions. The ratios of LnbY to denatured LnbX are 0 (filled circles), 1 (filled squares), 5 (open diamonds), 20 (open triangles), and 50 (filled triangles).