Biochemical Studies Of Galactokinase And UDP-galactose 4-epimerase

GalK,GalE1 and GalE2 are the key enzyme in the synthesis of Streptococcus pneumoniae capsule which is the important component of Streptococcus pneumoniae. The synthesis of GalK,GalE1 and GalE2 is coded by TIGR4 genome(GenBank accession no.NC₀03028). GalK,GalE1 and GalE2 are the key enzyme in the synthesis of Streptococcus pneumoniae capsule, they catalyse the synthesis of UDP-Gal. Capsule is the basis of host immune response, it can protect Streptococcus pneumoniae not to be identified by the host immune system in pathogen infection process.Protein and polysaccharide of capsule have been researched in therapy, but only polysaccharide of capsule can not induce immunological memory. Covalently link the polysaccharide to carrier protein is one of the forefront of drug development.Galactokinase(GalK) was cloned from the genome of Streptococcus pneumoniae 4 and was over-expressed in E. coli BL21 (DE3). The recombinant his-tagged GalK protein was purified by Ni-NTA column and gel filtration column.SDS-PAGE showed that the purity of GalK protein was more than 90%, the apparent molecular weight of the monomer of GalK protein was about 37 kDa, consistent with the calculated mass.The biochemical study showed that the optimal pH value of GalK protein was 7.5 and the optimal temperature was 45℃. ATP is not the only phosphate-donor of GalK protein. The reaction rate form Gal to Gal-1-P by GalK protein is 100%,3,5-dinitro-salicylic acid was ued to analyse and calculate the reaction rate of monosaccharide catalysed by GalK wild type and 4 mutant strains 176, 1764,17642 and 176423.3 mutant strains 1764,17642 and 176423 can use glucose as the substrate, but GalK wide type can not use it. Reaction was catalyzed by GalK mutant strains 176423,product Glc-1-P was identified by DNS and TLC, the purified Glc-1-P was identified by ESI-MS. ATP and N-acetylglucosamin or N-acetylgalactosamin or Fucose or Arabinose or Rhamnose were used as substrate, product was identified by DNS, TLC and MALDI-TOF or 1H-NMR or ESI-MS. When phosphate donor was CTP or dATP or dTTP or dCTP or dUTP, and acceptor was galactose, product Gal-1-P was analysed by DNS and TLC, identified by ESI-MS.UDP-galactose 4-epimerase (GalE) was cloned from the genome of Streptococcus pneumoniae 4 and was over-expressed in E. coli BL21 (DE3). The recombinant his-tagged GalE1 and GalE2 protein was purified by Ni-NTA column and gel filtration column.SDS-PAGE showed that the purity of GalE1 and GalE2 protein was more than 90%, the apparent molecular weight of the monomer of GalE1 and GalE2 protein was about 37 kDa, consistent with the calculated mass. The biochemical study showed that the optimal pH value of GalE1 and GalE2 protein was 8.0 and the optimal temperature was 37℃.UDP-Gal, UDP-GalNAc, UDP-Glc, UDP-GlcNAc were used as substrate to analyze the substrate specificity of GalE1 and GalE2, product was identified by capillary electrophoresis(CE).