Overexpression, one-step purification and characterization of UDP-glucose dehydrogenase and UDP-N-acetylglucosamine pyrophosphorylase.
Two enzymes of the Leloir pathway, UDP-GlcNAc pyrophosphorylase and UDP-Glc dehydrogenase, which are involved in the synthesis of activated sugar nucleotides have been cloned, overexpressed in Escherichia coli, and purified to homogeneity in only one step by chelation-affinity chromatography. The gene KfaC of E. coli K5 was thus demonstrated to encode UDP-Glc DH. Some properties of the cloned enzymes, such as stability, pH dependence, and substrate kinetics, were studied in order to facilitate the use of these enzymes in carbohydrate synthesis, especially in the synthesis of hyaluronic acid.
De Luca
C
C
Department of Chemistry, Scripps Research Institute, La Jolla, CA 92037, USA.
Lansing
M
M
Crescenzi
F
F
Martini
I
I
Shen
G J
GJ
O'Regan
M
M
Wong
C H
CH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D014533:Uridine Diphosphate Glucose Dehydrogenase; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase