The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 Å resolution

✍ Scribed by Alan J. Bauer; Ivan Rayment; Perry A. Frey; Dr. Hazel M. Holden

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: English
• Weight: 857 KB
• Volume: 12
• Category: Article
• ISSN: 0887-3585
• DOI: 10.1002/prot.340120409

✦ Synopsis

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 P2~1~2~1~2~1~ 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.