title: 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.
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Haruko
authors.initials: H
authors.lastname: Sakurama
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Masashi
authors.initials: M
authors.lastname: Kiyohara
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Jun
authors.initials: J
authors.lastname: Wada
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Honda
authors.affiliation: the Department of Organic Chemistry, Wakayama University, Sakaedani, Wakayama, 640-8510.
authors.forename: Masanori
authors.initials: M
authors.lastname: Yamaguchi
authors.affiliation: the Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589.
authors.forename: Satoru
authors.initials: S
authors.lastname: Fukiya
authors.affiliation: the Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yokota
authors.affiliation: the Faculty of Biology-Oriented Science and Technology, Kinki University, Kinokawa, Wakayama 649-6493, and.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Ashida
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Kumagai
authors.affiliation: the National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642, Japan.
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836.
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: From the Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836,. Electronic address: takane@ishikawa-pu.ac.jp.
authors.forename: Takane
authors.initials: T
authors.lastname: Katayama
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D005798:Genes, Bacterial / Q000502:physiology; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007223:Infant; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D006026:Glycoside Hydrolases; C076748:lacto-N-biosidase; D008274:Magnesium; D002118:Calcium
keywords: Bacterial Metabolism; Carbohydrate Metabolism; Glycobiology; Glycoside Hydrolases; Microbiology; Oligosaccharide