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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.
Highlights
Lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase
BLLJ_1506 alone failed to rescue the LNBaseϪ phenotype (Fig. 2, lane 6); when both BLLJ_1505 and BLLJ_1506 were introduced, the activity resumed (Fig. 2, lane 7). These results indicate that LNBase of B. longum JCM1217 consists of BLLJ_1505 and BLLJ_1506
The addition of purified LnbY to purified LnbX produced no effect on LNBase activity. These results indicated that the active LNBase molecule from B. longum is a single gene product
Summary
Lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Among the different HMO structures, four molecular species, namely 2Ј-fucosyllactose (Fuc␣1–2Gal1– 4Glc), lacto-N-tetraose (LNT, Gal1–3GlcNAc1–3Gal1– 4Glc), lacto-N-fucopentaose I (LNFP I, Fuc␣1–2Gal1–3GlcNAc1–3Gal1– 4Glc), and lactoN-difucohexaose I (Fuc␣1–2Gal1–3(Fuc␣1– 4)GlcNAc1–3Gal1– 4Glc), are most abundantly present and comprise more than 50% of the total oligosaccharides, unless the milk is derived from non-secretor or Lewis-negative subjects [9, 10] The latter three structures reveal an abundance of type-1 chains in the HMOs (see supplemental Fig. S1). The GH20 enzyme of B. longum JCM1217 was recently found to be -N-acetylglucosaminidase active on lacto-N-triose II (GlcNAc1–3Gal1– 4Glc) and on chitin oligosaccharides but not on LNT [29] These findings prompted us to identify a potentially novel LNBase gene encoded in the genome of B. longum. BLLJ_1505 and BLLJ_1506 homologs were found in the genomes of B. longum, B. bifidum, and a few gut microbes, suggesting that this LNBase has uniquely evolved in particular species
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