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

• Published in: The Journal of biological chemistry Vol. 288; no. 35; pp. 25194 - 25206
• Main Authors: Sakurama, Haruko, Kiyohara, Masashi, Wada, Jun, Honda, Yuji, Yamaguchi, Masanori, Fukiya, Satoru, Yokota, Atsushi, Ashida, Hisashi, Kumagai, Hidehiko, Kitaoka, Motomitsu, Yamamoto, Kenji, Katayama, Takane
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.08.2013; American Society for Biochemistry and Molecular Biology
• Subjects: Bacterial Metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Bifidobacterium - enzymology; Bifidobacterium - genetics; Calcium - chemistry; Calcium - metabolism; Carbohydrate Metabolism; Genes, Bacterial - physiology; Glycobiology; Glycobiology and Extracellular Matrices; Glycoside Hydrolases; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Glycoside Hydrolases - metabolism; Humans; Infant; Magnesium - chemistry; Magnesium - metabolism; Microbiology; Oligosaccharide; Oligosaccharides - chemistry; Oligosaccharides - genetics; Oligosaccharides - metabolism; Substrate Specificity; Glycobiology; Glycoside Hydrolases; Microbiology; Bacterial Metabolism; Oligosaccharide; Carbohydrate Metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.484733

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 (Ca2+ and Mg2+) 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. Background: Phenotypically lacto-N-biosidase-positive Bifidobacterium longum JCM1217 does not possess a gene homologous to previously identified lacto-N-biosidase. Results: Hypothetical proteins BLLJ_1505 and BLLJ_1506 encode lacto-N-biosidase and its designated chaperone, respectively. Conclusion: The enzyme showed unique and unexpected substrate specificity. Significance: The enzyme is important for understanding how B. longum consumes human milk oligosaccharides and also may serve as a new tool in glycobiology.