Identification of Lacto- N -Biose I Phosphorylase from Vibrio vulnificus CMCP6

Journal of Biological Chemistry

et al. 2009

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. GLNBP homologues are often found in human pathogenic and commensal bacteria, and their substrate specificities potentially define the nutritional acquisition ability of these microbes in their habitat. We report the crystal structures of GLNBP in five different ligand-binding forms. This is the first three-dimensional structure of glycoside hydrolase (GH) family 112. The GlcNAc-and GalNAc-bound forms provide structural insights into distinct substrate preferences of GLNBP and its homologues from pathogens. The catalytic domain consists of a partially broken TIM barrel fold that is structurally similar to a thermophilic ␤-galactosidase, strongly supporting the current classification of GLNBP homologues as one of the GH families. Anion binding induces a large conformational change by rotating a half-unit of the barrel. This is an unusual example of molecular adaptation of a TIM barrel scaffold to substrates.

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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The Crystal Structure of Galacto-N-biose/Lacto-N-biose I Phosphorylase

Hidaka

Journal of Biological Chemistry

et al. 2009

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“…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.…”

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confidence: 99%

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

Nakajima

Bioscience, Biotechnology, and Biochemistry

2010

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D-Galactosyl-1!4-L-rhamnose (GalRha) was produced enzymatically from 1.1 M sucrose and 1.0 M L-rhamnose by the concomitant actions of four enzymes (sucrose phosphorylase, UDP-glucose-hexose 1-phosphate uridylyltransferase, UDP-glucose 4-epimerase, and D-galactosyl-1!4-L-rhamnose phosphorylase) in the presence of 1.0 mM UDP-glucose and 30 mM inorganic phosphate. The accumulation of GalRha in 1 liter of the reaction mixture reached 230 g (the reaction yield was 71% from L-rhamnose). Sucrose and fructose in the reaction mixture were removed by yeast treatment, but isolation of GalRha by crystallization after yeast treatment was unsuccessful. Finally, 49 g of GalRha was isolated from part of the reaction mixture with yeast treatment by gel-filtration chromatography.

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

Nakajima

Appl Microbiol Biotechnol

2009

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.