High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.
UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 A resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.
Thoden
J B
JB
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705, USA.
Frey
P A
PA
Holden
H M
HM
D015089:20-Hydroxysteroid Dehydrogenases / Q000737:chemistry; D018360:Crystallography, X-Ray; D004093:Dihydropteridine Reductase / Q000737:chemistry; D009243:NAD / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010636:Phenols / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*
D010636:Phenols; D011994:Recombinant Proteins; D009243:NAD; D015089:20-Hydroxysteroid Dehydrogenases; D004093:Dihydropteridine Reductase; D014534:UDPglucose 4-Epimerase; C020551:galactose epimerase