Structural and kinetic studies of sugar binding to galactose mutarotase from Lactococcus lactis.
Galactose mutarotase catalyzes the conversion of beta-D-galactose to alpha-D-galactose in the Leloir pathway for galactose metabolism. The high resolution x-ray structure of the dimeric enzyme from Lactococcus lactis was recently solved and shown to be topologically similar to the 18-stranded, anti-parallel beta-motif observed for domain 5 of beta-galactosidase. In addition to determining the overall molecular fold of galactose mutarotase, this initial investigation also provided a detailed description of the electrostatic interactions between the enzyme and its physiologically relevant substrate, galactose. Specifically, the side chains of His-96 and His-170 were shown to be located within hydrogen bonding distance to the C-5 oxygen of the substrate, while the carboxylate of Glu-304 was positioned near the C-1 hydroxyl group of the sugar. On the basis of this initial study, a possible role for Glu-304 as the general acid/base group in catalysis was put forth. Here we describe the combined x-ray crystallographic and kinetic analyses of L. lactis galactose mutarotase complexed with D-glucose, D-fucose, D-quinovose, L-arabinose, or D-xylose. These investigations have revealed that there are several distinct binding modes for these sugars, which are dependent upon the spatial orientation of the C-4 hydroxyl group. In those sugars with the same C-4 hydroxyl group orientation as galactose, their C-1 hydroxyl groups are invariably located near Glu-304. For those sugars, which have the same C-4 hydroxyl group configuration as glucose, the C-1 hydroxyls are typically located near Asp-243. These different binding modes correlate with both the observed kinetic parameters and the presence or absence of a hydrogen bond between the guanidinium group of Arg-71 and the C-4 hydroxyl group of the sugar ligand.
Thoden
James B
JB
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706-1544, USA. JBThoden@facstaff.wisc.edu
Kim
Jungwook
J
Raushel
Frank M
FM
Holden
Hazel M
HM
D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005690:Galactose / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D013294:Lactococcus lactis / Q000201:enzymology*; D008958:Models, Molecular; D015394:Molecular Structure; D009005:Monosaccharides / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D009005:Monosaccharides; D002238:Carbohydrate Epimerases; C459183:galactose mutarotase; D005690:Galactose