Characterization of β-1,3-galactosyl-N-acetylhexosamine phosphorylase from Propionibacterium acnes

Abstract

Homologs of the beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalHexNAcP) gene (gnpA) were cloned from the genomic DNA of Propionibacterium acnes JCM6425 and P. acnes JCM6473, showing 99.9% and 97.9% nucleotide sequence identity, respectively, with the ppa0083 gene from the genome-sequenced P. acnes KPA171202. No gnpA gene was detected in the genomic DNA of type strain P. acnes ATCC25746. The recombinant enzyme from P. acnes JCM6425 (GnpA) showed approximately 70 times higher specific activity of phosphorolysis on galacto-N-biose (Galbeta1-->3GalNAc, GNB) than that on lacto-N-biose I (Galbeta1-->3GlcNAc). K (m) value for GnpA on GNB was high, but GnpA did not exhibit activity on any derivatives of GNB examined. These results indicate that GnpA is GalHexNAcP which should be classified as galacto-N-biose phosphorylase. The large k (cat) value of GnpA on GalNAc suggests that GnpA would be a useful catalyst for the synthesis of GNB.

Characterization of Three β-Galactoside Phosphorylases from Clostridium phytofermentans

“…To date, GalHexNAcP is the only phosphorylase known to act on ␤-galactoside. This enzyme was first found in the cell-free extract of Bifidobacterium bifidum (14) and then in B. longum (1,15), Clostridium perfringens (16), Propionibacterium acnes (17), and Vibrio vulnificus (18). These studies revealed that GalHexNAcPs were classified into three subgroups based on substrate preference between GNB and LNB.…”

Unique Sugar Metabolic Pathways of Bifidobacteria

“…The GalHexNAcPs from C. perfringens ATCC13124 and Propionibacterium acnes JCM6425 are specific for GNB, and they are named GNB phosphorylase. 98,99) Since C. perfringens strains usually colonize the human GIT along with bifidobacteria, the inability of this pathogen to utilize LNB is an feature important to the prebiotic effect of LNB. P. acnes is a skin-commensal bacterium that causes skin diseases such as acne.…”

Practical Preparation ofD-Galactosyl-β1→4-L-rhamnose Employing the Combined Action of Phosphorylases

“…The concentration of Pi was measured as previously described. 16) The K values were calculated using the initial substrate concentrations and the Pi concentration in equilibrium.…”

“…The results are given in Table 1. The K values for the syntheses of LNB and GNB 16) were significantly higher than that of GalRha, suggesting that the difference in yield is explainable by the difference in the equilibria of the phosphorolyses. The difference in equilibrium cannot be explained by the difference in the 1!3 and 1!4 linkages, because the K value of GalRha was close to that of D-galactosyl-1!3-D-glucose.…”

“…9,10) This enzyme was found among homologs of GalHexNAcP, but the substrate specificity of the enzyme is completely different from those of known GalHexNAcPs. [11][12][13][14][15][16] GalRhaP is the only phosphorylase acting on Rha as an acceptor among phosphorylases to date.…”