Preparation and characterization of a bifunctional fusion enzyme composed of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase.
A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid. The fusion enzyme was expressed in E. coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose. On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities. The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes. The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.
Tamada
Y
Y
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
Swanson
B A
BA
Arabshahi
A
A
Frey
P A
PA
D000409:Alanine / Q000235:genetics; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005695:Galactosephosphates / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins; D013379:Substrate Specificity; D014534:UDPglucose 4-Epimerase / Q000235:genetics / Q000378:metabolism*; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D005695:Galactosephosphates; D005958:Glucosephosphates; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C029973:galactose-1-phosphate; D014531:Uridine Diphosphate Galactose; C031590:glucose-1-phosphate; D005694:UTP-Hexose-1-Phosphate Uridylyltransferase; D014534:UDPglucose 4-Epimerase; D000409:Alanine; D014532:Uridine Diphosphate Glucose