Characterization of three beta-galactoside phosphorylases from Clostridium phytofermentans: discovery of d-galactosyl-beta1->4-l-rhamnose phosphorylase.
We characterized three d-galactosyl-beta1-->3-N-acetyl-d-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-beta1-->3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-beta1-->3-d-GlcNAc), thus indicating that they are d-galactosyl-beta1-->3-N-acetyl-d-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using alpha-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-beta1-->4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were beta-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-beta1-->4-l-rhamnose were k(cat) = 45 s(-1) and K(m) = 7.9 mm, thus indicating that these values are common among other phosphorylases. We propose d-galactosyl-beta1-->4-l-rhamnose phosphorylase as the name for Cphy1920 protein.
Nakajima
Masahiro
M
National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Nishimoto
Mamoru
M
Kitaoka
Motomitsu
M
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002851:Chromatography, High Pressure Liquid; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005697:Galactosides / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D006005:Phosphorylases / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D012210:Rhamnose / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D005697:Galactosides; D007527:Isoenzymes; C007816:beta-galactoside; D006005:Phosphorylases; D012210:Rhamnose