Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124

  • 206 Accesses

  • 26 Citations


Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galβ1→3GlcNAc, LNB) and galacto-N-biose (Galβ1→3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of α-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a k cat/K m value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar k cat/K m values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-α-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Aminoff D, Furukawa K (1970) Enzymes that destroy blood group specificity. J Biol Chem 245:1659–1669

  2. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

  3. Benno Y, Sawada K, Mitsuoka T (1984) The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants. Microbiol Immun 28:975–986

  4. Canard B, Garnier T, Saintjoanis B, Cole ST (1994) Molecular genetic analysis of the NagH gene encoding a hyaluronidase of Clostridium perfringens. Mol Gen Genet 243:215–224

  5. Derensy-Dron D, Krzewinski F, Brassart C, Bouquelet S (1999) β-1,3-Galactosyl-N-acetylhexosamine phosphorylase from Bifidobacterium bifidum DSM 20082: characterization, partial purification and relation to mucin degradation. Biotechnol Appl Biochem 29:3–10

  6. Ficko-Blean E, Boraston AB (2006) The interaction of a carbohydrate-binding module from a Clostridium perfringensN-acetyl-β-hexosaminidase with its carbohydrate receptor. J Biol Chem 281:37748–37757

  7. Fujita K, Oura F, Nagamine N, Katayama T, Hiratake J, Sakata K, Kumagai H, Yamamoto K (2005) Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-α-N-acetylgalactosaminidase from Bifidobacterium longum. J Biol Chem 280:37415–37422

  8. Honda Y, Kitaoka M, Hayashi K (2004) Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio. Biochem J 377:225–232

  9. Ishiyama N, Creuzenet C, Lam JS, Berghuis AM (2004) Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa—substrate specificity in UDP-hexose 4-epimerases. J Biol Chem 279:22635–22642

  10. Katayama T, Sakuma A, Kimura T, Makimura Y, Hiratake J, Sakata K, Yamanoi T, Kumagai H, Yamamoto K (2004) Molecular cloning and characterization of Bifidobacterium bifidum 1,2-α-L-fucosidase (AfcA), a novel inverting glycosidase (Glycoside hydrolase family 95). J Bacteriol 186:4885–4893

  11. Kim YK, Kitaoka M, Krishnareddy M, Mori Y, Hayashi K (2002) Kinetic studies of a recombinant cellobiose phosphorylase (CBP) of the Clostridium thermocellum YM4 strain expressed in Escherichia coli. J Biochem 132:197–203

  12. Kitaoka M, Sasaki T, Taniguchi H (1992) Phosphorolytic reaction of Cellvibrio gilvus cellobiose phosphorylase. Biosci Biotechnol Biochem 56:652–655

  13. Kitaoka M, Sasaki T, Taniguchi H (1993) Purification and properties of laminaribiose phosphorylase (EC from Euglena gracilis Z. Arch Biochem Biophys 304:508–514

  14. Kitaoka M, Tian J, Nishimoto M (2005) Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum. Appl Environ Microbiol 71:3158–3162

  15. Lievin V, Peiffer I, Hudault S, Rochat F, Brassart D, Neeser JR, Servin AL (2000) Bifidobacterium strains from resident infant human gastrointestinal microflora exert antimicrobial activity. Gut 47:646–652

  16. Lowry OH, Lopez JA (1946) The determination of inorganic phosphate in the presence of labile phosphate esters. J Biol Chem 162:421–428

  17. Matsumoto M, Tani H, Ono H, Ohishi H, Benno Y (2002) Adhesive property of Bifidobacterium lactis LKM512 and predominant bacteria of intestinal microflora to human intestinal mucin. Curr Microbiol 44:212–215

  18. Matsushita O, Yoshihara K, Katayama SI, Minami J, Okabe A (1994) Purification and characterization of a Clostridium perfringens 120-kilodalton collagenase and nucleotide sequence of the corresponding gene. J Bacteriol 176:149–156

  19. McDonel JL (1980) Clostridium perfringens toxins (type A, B, C, D, E). Pharmacol Ther 10:617–655

  20. Myers GSA, Rasko DA, Cheung JK, Ravel J, Seshadri R, DeBoy RT, Ren QH, Varga J, Awad MM, Brinkac LM, Daugherty SC, Haft DH, Dodson RJ, Madupu R, Nelson WC, Rosovitz MJ, Sullivan SA, Khouri H, Dimitrov GI, Watkins KL, Mulligan S, Benton J, Radune D, Fisher DJ, Atkins HS, Hiscox T, Jost BH, Billington SJ, Songer JG, McClane BA, Titball RW, Rood JI, Melville SB, Paulsen IT (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16:1031–1040

  21. Nidetzky B, Eis C, Albert M (2000) Role of non-covalent enzyme-substrate interactions in the reaction catalysed by cellobiose phosphorylase from Cellulomonas uda. Biochem J 351:649–659

  22. Nielsen H, Engelbrecht J, Brunak S, vonHeijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6

  23. Nihira T, Nakajima M, Inoue K, Kitaoka M (2007) Colorimetric quantification of α-galactose 1-phosphate. Anal Biochem 371:259–261

  24. Nishimoto M, Kitaoka M (2007a) The complete lacto-N-biose I/galacto-N-biose metabolic pathway in Bifidobacterium longum: identification of N-acetylhexosamine 1-kinase. Appl Environ Microbiol 73:6444–6449

  25. Nishimoto M, Kitaoka M (2007b) Identification of the putative proton donor residue of lacto-N-biose phosphorylase (EC Biosci Biotechnol Biochem 71:1587–1591

  26. Nishimoto M, Kitaoka M (2007c) Practical preparation of Lacto-N-biose I, the candidate of the bifidus factor in human milk. Biosci Biotechnol Biochem 71:2101–2104

  27. Petit L, Gibert M, Popoff MR (1999) Clostridium perfringens: toxinotype and genotype. Trends Microbiol 7:104–110

  28. Petuely F (1957) Bifidusflora bei Fraschenkindern durch bifidogene Substanzen (Bifidusfaktor). Z Kinderheilkd 79:174–179

  29. Rajashekhara E, Kitaoka M, Kim YK, Hayashi K (2002) Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8. Biosci Biotechnol Biochem 66:2578–2586

  30. Rood JI, Cole ST (1991) Molecular genetics and pathogenesis of Clostridium perfringens. Microbiol Rev 55:621–648

  31. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99:996–1001

  32. Titball RV, Naylor CE, Basak AK (1999) The Clostridium perfringens α-toxin. Anaerobe 5:51–64

  33. Tsumuraya Y, Brewer CF, Hehre EJ (1990) Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of α-maltose and α-D-glucose controls the enzymes glucosyltransferase activity. Arch Biochem and Biophys 281:58–65

  34. Young PR, Snyder WR, McMahon RF (1991) Kinetic mechanism of Clostridium perfringens phospholipase C. Biochem J 280:407–410

Download references


This work was supported in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).

Author information

Correspondence to Motomitsu Kitaoka.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Nakajima, M., Nihira, T., Nishimoto, M. et al. Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124. Appl Microbiol Biotechnol 78, 465–471 (2008).

Download citation


  • EC
  • Galacto-N-biose phosphorylase
  • Galacto-N-biose
  • Clostridium perfringens
  • Lacto-N-biose I
  • Mucin