Structural and Kinetic Studies of Sugar Binding to Galactose Mutarotase from Lactococcus lactis

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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...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular Structure of Human Galactose Mutarotase

Thoden

Timson

Reece

et al. 2004

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“…Although galactosemia is rare, it is a potentially lethal genetic disease with clinical manifestations, including intellectual retardation, liver dysfunction, and cataract formation, among others. At present, the high resolution x-ray crystallographic structures of three of the four enzymes in the pathway, namely galactose mutarotase, galactose-1-phosphate uridylyltransferase, and UDP-galactose 4-epimerase, are known through efforts by this laboratory (3)(4)(5)(6)(7)(8)(9). The remaining enzyme of unknown structure, galactokinase, catalyzes the second step of the pathway, the ATP-dependent conversion of ␣-D-galactose to galactose 1-phosphate.…”

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confidence: 99%

Molecular Structure of Galactokinase

Thoden

Holden

2003

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Galactokinase plays a key role in normal galactose metabolism by catalyzing the ATP-dependent phosphorylation of ␣-D-galactose to galactose 1-phosphate. In humans, mutations in the galactokinase gene can lead to the diseased state referred to as Type II galactosemia. Here we describe the three-dimensional structure of galactokinase from Lactococcus lactis determined to 2.1-Å resolution. As expected from amino acid sequence alignments, galactokinase adopts a similar topology to that observed for members of the GHMP superfamily. The N-terminal domain is characterized by a five-stranded mixed ␤-sheet while the C-terminal motif is dominated by two distinct four-stranded anti-parallel ␤-sheets. is positioned within 3.5 Å of the C-1 hydroxyl group of galactose, whereas the guanidinium group of Arg 36 is situated between both the C-1 hydroxyl group and the inorganic phosphate. Most likely these residues play key roles in catalysis. The structure of galactokinase described here serves as a model for understanding the functional consequences of point mutations known to result in Type II galactosemia in humans.

“…This pocket, which contains a glycerol-bound molecule bound in the so-called apo structure, corresponds to the active site in galactose mutarotase. Crystal structures of Lactococcus lactis galactose mutarotase bound to sugars have shown that these residues are involved in sugar binding (11,25). Site-directed mutagenesis led to the conclusion that in L. lactis galactose mutarotase, His 170 and Glu 304 play the role of catalytic acid and base, respectively (26).…”

Section: Resultsmentioning

confidence: 99%

Structure-based Functional Annotation

Graille

Baltaze

Leulliot

et al. 2006

Despite the generation of a large amount of sequence information over the last decade, more than 40% of well characterized enzymatic functions still lack associated protein sequences. Assigning protein sequences to documented biochemical functions is an interesting challenge. We illustrate here that structural genomics may be a reasonable approach in addressing these questions. We present the crystal structure of the Saccharomyces cerevisiae YMR099cp, a protein of unknown function. YMR099cp adopts the same fold as galactose mutarotase and shares the same catalytic machinery necessary for the interconversion of the ␣ and ␤ anomers of galactose. The structure revealed the presence in the active site of a sulfate ion attached by an arginine clamp made by the side chain from two strictly conserved arginine residues. This sulfate is ideally positioned to mimic the phosphate group of hexose 6-phosphate. We have subsequently successfully demonstrated that YMR099cp is a hexose-6-phosphate mutarotase with broad substrate specificity. We solved high resolution structures of some substrate enzyme complexes, further confirming our functional hypothesis. The metabolic role of a hexose-6-phosphate mutarotase is discussed. This work illustrates that structural information has been crucial to assign YMR099cp to the orphan EC activity: hexose-phosphate mutarotase.