Molecular Structure of Human Galactose Mutarotase

Abstract

Galactose mutarotase catalyzes the conversion of ␤-Dgalactose to ␣-D-galactose during normal galactose metabolism. The enzyme has been isolated from bacteria, plants, and animals and is present in the cytoplasm of most cells. Here we report the x-ray crystallographic analysis of human galactose mutarotase both in the apoform and complexed with its substrate, ␤-D-galactose. The polypeptide chain folds into an intricate array of 29 ␤-strands, 25 classical reverse turns, and 2 small ␣-helices. There are two cis-peptide bonds at Arg-78 and Pro-103. The sugar ligand sits in a shallow cleft and is surrounded by Asn-81, Arg-82, His-107, His-176, Asp-243, Gln-279, and Glu-307. Both the side chains of Glu-307 and His-176 are in the proper location to act as a catalytic base and a catalytic acid, respectively. These residues are absolutely conserved among galactose mutarotases. To date, x-ray models for three mutarotases have now been reported, namely that described here and those from Lactococcus lactis and Caenorhabditis elegans. The molecular architectures of these enzymes differ primarily in the loop regions connecting the first two ␤-strands. In the human protein, there are six extra residues in the loop compared with the bacterial protein for an approximate longer length of 9 Å. In the C. elegans protein, the first 17 residues are missing, thereby reducing the total number of ␤-strands by one.During normal galactose metabolism, ␤-D-galactose is converted to glucose 1-phosphate via the action of four enzymes that constitute the Leloir pathway as shown in Scheme 1 (1). In the first step of this pathway, ␤-D-galactose is epimerized to ␣-D-galactose through the action of galactose mutarotase. The second step involves the phosphorylation of ␣-D-galactose to galactose 1-phosphate by galactokinase. As indicated in Scheme 1, galactose 1-phosphate uridylyltransferase catalyzes the third step by transferring a UMP group from UDP-glucose to galactose 1-phosphate, thereby generating glucose 1-phosphate and UDP-galactose. To complete the pathway, UDPgalactose is converted to UDP-glucose by UDP-galactose 4-epimerase.Mutations in three of the enzymes of the Leloir pathway, namely galactokinase, galactose 1-phosphate uridylyltransferase, or UDP-galactose 4-epimerase, have been demonstrated to result in the diseased state known as galactosemia (2, 3). The symptoms of this genetic disease include early onset cataracts (typically within the first two years of life) and, in more severe cases, liver, kidney, and brain damage. Cataract formation is believed to be caused by the buildup of unmetabolized galactose in the lens of the eye. Aldose reductase catalyzes the reduction of the sugar to galactitol (dulcitol), which is not, in contrast to galactose, readily transported across the plasma membrane (4 -6). As such, the accumulation of this highly osmotically active compound draws excess water into the lens resulting in cataracts by a mechanism that is not fully understood. The more severe symptoms of galactosemia are most likely caused b...

Structure-based Functional Annotation

“…The two helices ␣1 and ␣2 are facing toward the same side of the monomer and are part of the linkers connecting strands ␤7 to ␤8 and ␤19 to ␤20. This fold has already been described for galactose mutarotases (RMSD of 2.2-2.6 Å over 240 C␣ positions; 15-17% sequence identity (11,20,21)), domain 5 of ␤-galactosidase (RMSD of 3.1 Å over 190 C␣ positions, 7% sequence identity (22)) and the N-terminal domain of maltose phosphorylase (RMSD of 3.4 Å over 164 C␣ positions, 13% sequence identity (23)). Structural similarity is also shared with proteins of unknown function: Caenorhabditis elegans C01B4.6 gene product (RMSD of 2.41 Å over 235 C␣ positions, 13% sequence identity; PDB code: 1LUR) and S. cerevisiae YNR071cp (RMSD of 2.67 Å over 243 C␣ positions, 12% sequence identity; PDB code: 1YGA).…”

“…A two-layered ␤-sandwich fold made of 20 -30 antiparallel ␤-strands was revealed for L. lactis, human and yeast enzymes (11,20,21). In addition, the structure of the galactosebound enzyme has highlighted four strictly conserved residues (2 histidines and two acidic amino acids) located in the galactose binding pocket that could potentially act as general acid/ base in the mutarotation reaction.…”

Galactokinase promiscuity: a question of flexibility?

“…Longer-term symptoms include speech impairments, cognitive disabilities, female reproductive Representative structures of the enzymes are shown next to the corresponding reactions. The human structures of galactose mutarotase (GALM, 1SO0 [74]), GALK1 (1WUU [42]) and GALE (1EK6 [75]) are shown. Since there is currently no structure of the human GALT known, the E. coli structure (1HXQ [76]) is shown for this enzyme.…”

The structure of the first representative of Pfam family PF06475 reveals a new fold with possible involvement in glycolipid metabolism

“…; Thoden et al, 2004) and meso-2,3-butanediol dehydrogenase (PDB code 1geg; 20 atoms in common, Tanimoto similarity score 0.36, Z score 3.82, P value 1.32Â 10 À2 ; Otagiri et al, 2001) as the closest matches, in addition to an inorganic pyrophosphatase (PDB code 1wpm; 25 atoms in common, Tanimoto similarity score 0.37, Z score 3.89, P value 1.21 Â 10…”