title: Biochemical characterization of a UDP-sugar pyrophosphorylase from Thermus caldophilus GK24.
abstract: An extremely thermostable UDP-GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion-exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8-fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel-filtration chromatography. The activity was maximum at 86 degrees C and its half-life at 95 degrees C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP-GlcNAc pyrophosphorylase activity could be enhanced by fructose 1-phosphate, a precursor of UDP-GlcNAc. The enzyme showed a broad substrate specificity with sugar 1-phosphates, including glucose 1-phosphate, GlcNAc-1-P and xylose 1-phosphate. The enzyme was therefore named UDP-sugar pyrophosphorylase. The N-terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N-terminal sequence is similar to that of UDP-GlcNAc and UDP-glucose pyrophosphorylases from other bacterial sources.
authors.affiliation: Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Yusong, Taejon, 305-600, South Korea.
authors.forename: J S
authors.initials: JS
authors.lastname: Kim
authors.forename: S
authors.initials: S
authors.lastname: Koh
authors.forename: H J
authors.initials: HJ
authors.lastname: Shin
authors.forename: D S
authors.initials: DS
authors.lastname: Lee
authors.forename: S Y
authors.initials: SY
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase