The molecular structure of UDP‐galactose 4‐epimerase from Escherichia coli determined at 2.5 Å 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 Å. 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 P2₁2₁2₁ with unit cell dimensions of a = 76.3 Å, b = 83.1 Å, c = 132.1 Å, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 Å 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 β-pleated sheet flanked on either side of α-helices. The seventh strand of the β-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 β-pleated sheet, two major α-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.