title: The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution.
abstract: UDP-galactose 4-epimerase catalyzes the conversion of UDP-galactose to UDP-glucose during normal galactose metabolism. The molecular structure of UDP-galactose 4-epimerase from Escherichia coli has now been solved to a nominal resolution of 2.5 A. As isolated from E. coli, the molecule is a dimer of chemically identical subunits with a total molecular weight of 79,000. Crystals of the enzyme used for this investigation were grown as a complex with the substrate analogue, UDP-benzene, and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 76.3 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A resolution was obtained by a combination of multiple isomorphous replacement with six heavy atom derivatives, molecular averaging, and solvent flattening. Each subunit of epimerase is divided into two domains. The larger N-terminal domain, composed of amino acid residues 1-180, shows a classic NAD+ binding motif with seven strands of parallel beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-pleated sheet is contributed by amino acid residues from the smaller domain. In addition, this smaller C-terminal domain, consisting of amino acid residues 181-338, contains three strands of beta-pleated sheet, two major alpha-helices and one helical turn. The substrate analogue, UDP-benzene, binds in the cleft located between the two domains with its phenyl ring in close proximity to the nicotinamide ring of NAD+. Contrary to the extensive biochemical literature suggesting that epimerase binds only one NAD+ per functional dimer, the map clearly shows electron density for two nicotinamide cofactors binding in symmetry-related positions in the dimer. Likewise, each subunit in the dimer also binds one substrate analogue.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison 53705.
authors.forename: A J
authors.initials: AJ
authors.lastname: Bauer
authors.forename: I
authors.initials: I
authors.lastname: Rayment
authors.forename: P A
authors.initials: PA
authors.lastname: Frey
authors.forename: H M
authors.initials: HM
authors.lastname: Holden
mesh_terms: D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D014534:UDPglucose 4-Epimerase / Q000737:chemistry*; D014961:X-Ray Diffraction
chemical_list: D014534:UDPglucose 4-Epimerase