Sugar Nucleotide Pyrophosphorylases from Thermus caldophilus GK24
Joongsu KIM, Sukhoon KOH, Mikyung KIM, and Dae-Sil LEE
Molecular Glycobiology Research Unit, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Taejeon, South Korea
In studying biosynthesis of the bacterial carbohydrates, a number of sugar nucleotide pyrophosphorylases from Thermus caldophilus GK24 have been searched through HPLC analysis. It allowed to identify ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, UDP-N-Acetylglucosamine pyrophosphorylase, UDP-xylose pyrophosphorylase, GDP-mannose pyrophosphorylase and UDP-mannose pyrophosphorylase. At first, ADP-glucose pyrophosphorylase has been purified, and biochemically and genetically characterized, including production of recombinant ADP-glucose pyrophosphorylase in E. coli. Secondly, UDP-N-Acetylglucosamine pyrophosphorylase was purified through chromatographical methods, using DEAE- sephacel, Phenyl-sepharose, blue-sepharose and Mono-Q. Based on its peptide sequences, primers were designed for PCR, which was used as a probe in Southern blot and colony hybridization for cloning the gene from cosmid library. Its molecular weight was 38 kDa, estimated from its gene sequence. The enzyme, which expressed in E. coli, was purified by heat treatment and DEAE-sephacel chromatography, and biochemically characterized in the presence of various hexose-1-phosphate. Interestingly, the enzyme has three different activities; UDP-xylose pyrophosphorylase, UDP-glucose pyrophosphorylase and UDP-N-Acetylglucosamine pyrophosphorylase. UDP-xylose pyrophosphorylase showed to be respectively 2 and 5 times higher than those of UDP-glucose pyrophosphorylase and UDP-N- Acetylglucosamine pyrophosphorylase. Hence, the enzyme was renamed as UDP- xylose pyrophosphorylase (UDPX). UDPX was reexamined in terms of activities, reaction temperature, allosteric effect, metal ions and kinetics.
