UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity.

UDPgalactose 4-epimerase (epimerase) catalyzes the reversible conversion between UDPgalactose and UDPglucose and is an important enzyme of the galactose metabolic pathway. The Saccharomyces cerevisiae epimerase encoded by the GAL10 gene is about twice the size of either the bacterial or human protein. Sequence analysis indicates that the yeast epimerase has an N-terminal domain (residues 1-377) that shows significant similarity with Escherichia coli and human UDPgalactose 4-epimerase, and a C-terminal domain (residues 378-699), which shows extensive identity to either the bacterial or human aldose 1-epimerase (mutarotase). The S. cerevisiae epimerase was purified to > 95% homogeneity by sequential chromatography on DEAE-Sephacel and Resource-Q columns. Purified epimerase preparations showed mutarotase activity and could convert either alpha-d-glucose or alpha-d-galactose to their beta-anomers. Induction of cells with galactose led to simultaneous enhancement of both epimerase and mutarotase activities. Size exclusion chromatography experiments confirmed that the mutarotase activity is an intrinsic property of the yeast epimerase and not due to a copurifying endogenous mutarotase. When the purified protein was treated with 5'-UMP and l-arabinose, epimerase activity was completely lost but the mutarotase activity remained unaffected. These results demonstrate that the S. cerevisiae UDPgalactose 4-epimerase is a bifunctional enzyme with aldose 1-epimerase activity. The active sites for these two enzymatic activities are located in different regions of the epimerase holoenzyme.     

The molecular architecture of galactose mutarotase/UDP-galactose 4-epimerase from Saccharomyces cerevisiae

The metabolic pathway by which beta-D-galactose is converted to glucose 1-phosphate is known as the Leloir pathway and consists of four enzymes. In most organisms, these enzymes appear to exist as soluble entities in the cytoplasm. In yeast such as Saccharomyces cerevisiae, however, the first and last enzymes of the pathway, galactose mutarotase and UDP-galactose 4-epimerase, are contained within a single polypeptide chain referred to as Gal10p. Here we report the three-dimensional structure of Gal10p in complex with NAD(+), UDP-glucose, and beta-D-galactose determined to 1.85-A resolution. The enzyme is dimeric with dimensions of approximately 91 A x 135 A x 108 A and assumes an almost V-shaped appearance. The overall architecture of the individual subunits can be described in terms of two separate N- and C-terminal domains connected by a Type II turn formed by Leu-357 to Val-360. The first 356 residues of Gal10p fold into the classical bilobal topology observed for all other UDP-galactose 4-epimerases studied thus far. This N-terminal domain contains the binding sites for NAD(+) and UDP-glucose. The polypeptide chain extending from Glu-361 to Ser-699 adopts a beta-sandwich motif and harbors the binding site for beta-D-galactose. The two active sites of Gal10p are separated by over 50 A. This investigation represents the first structural analysis of a dual function enzyme in the Leloir pathway.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis. Evidence for independent mutarotation site.

UDP-galactose 4-epimerases from the yeast Kluyvero-myces fragilis and Escherichia coli are both homodimers but the molecular mass of the former (75 kDa/subunit) is nearly double that of the latter (39 kDa/subunit). Protein databank sequence homology revealed the possibility of mutarotase activity in the excess mass of the yeast enzyme. This was confirmed by three independent assay protocols. With the help of specific inhibitors and chemical modification reagents, the catalytic sites of epimerase and mutarotase were shown to be distinct and independent. Partial proteolysis with trypsin in the presence of specific inhibitors, 5'-UMP for epimerase and galactose for mutarotase, protected the respective activities. Similar digestion with double inhibitors cleaved the molecule into two fragments of 45 and 30 kDa. After separation by size-exclusion HPLC, they manifested exclusively epimerase and mutarotase activities, respectively. Epimerases from Kluyveromyces lactis var lactis, Pachysolen tannophilus and Schizosaccharomyces pombi also showed associated mutarotase activity distinct from the constitutively formed mutarotase activity. Thus, the bifunctionality of homodimeric yeast epimerases of 65-75 kDa/subunit appears to be universal. In addition to the inducible bifunctional epimerase/mutarotase, K. fragilis contained a smaller constitutive monomeric mutarotase of approximately 35 kDa.     

Comparative modeling and genomics for galactokinase (Gal1p) enzyme

The Gal1p (Galactokinase) protein is known for regulation of D-galactose metabolism. It catalyzes the formation of galactose -1-phosphate from alpha - D-galactose, which is an important step in galactose catabolism. The knowledge of Gal1p protein structure, its protein interacting partners and enumeration of functional site residues will provide great insight in understanding the functional role of Gal1p. These studies are lacking in case of the Gal11p kinase enzyme. Structure of this enzyme has already been determined in S. cerevisiae, however, no structural information for this protein is available for K. lactis and E. coli. We used the homology modeling based approach to model the structures of Gal1p for K. lactis and E. coli. Furthermore, functional residues were predicted for these Gal1 proteins and the strength of interaction between Gal1p and other Gal proteins was determined by protein-protein interaction studies via patchdock software. The interaction studies revealed that the affinity for Gal1p for other Gal proteins varies in different organisms. Sequence and structural based comparison of Gal1p kinase enzyme showed that the orthologs in K.lactis and S. cervisiae are more similar to each other as compared to the ortholog in E. coli. These studies carried out by us will help in better understanding of the galactose metabolism. Our sequence and structure comparison studies revealed that Human Gal1p shows more homology for Gal1p protein of E. coli. The above studies may be applied to Human Gal1p, where it can help in gaining useful insight into Galactosemia disease.     

Galactose transporters discriminate steric anomers at the cell surface in yeast

Aldose-1-epimerase or mutarotase (EC 5.1.3.3) catalyzes interconversion of α/β-anomers of aldoses, such as glucose and galactose, and is distributed in a wide variety of organisms from bacteria to humans. Nevertheless, the physiological role of this enzyme has been elusive in most cases, because the α-form of aldoses in the solid state spontaneously converts to the β-form in an aqueous solution until an equilibrium of α : β=36.5 : 63.5 is reached. A gene named GAL10 encodes this enzyme in yeast. Here, we show that the GAL10 -encoded mutarotase is necessary for utilization of galactose in the milk yeast Kluyveromyces lactis , and that this condition is presumably created by the presence of the β-specific galactose transporter, which excludes the α-anomer from the α/β-mixture in the medium at the cell surface. Thus, we found that a mutarotase-deficient mutant of K. lactis failed to grow on medium, in which galactose was the sole carbon source, but, surprisingly, that the growth failure is suppressed by concomitant expression of the Saccharomyces cerevisiae -derived galactose transporter Gal2p, but not by that of the K. lactis galactose transporter Hgt1p. We also suggest the existence of another mutarotase in K. lactis , whose physiological role remains unknown, however.     

Structure modeling and comparative genomics for epimerase enzyme (Gal10p)

The Gal10p (UDP-Galactose 4-epimerase) protein is known for regulation of D-galactose metabolism. It catalyzes the inter-conversion between UDPgalactose and UDP-glucose. Knowledge of protein structure, neighboring interacting partners as well as functional residues of the Gal10p is crucial for carry out its function. These problems are still uncovered in case of the Epimerase enzyme. Structure of Epimerase enzyme has already been determined in S.cerevisiae and E.coli, however, no structural information for this protein is available for K.lactis. We used the homology modeling approach to model the structure of Gal10p in K.lactis. Furthermore, functional residues were predicted for modeled Gal10 protein and the strength of interaction between Gal10p and other Gal proteins was carried out by protein -protein interaction studies. The interaction studies revealed that the affinity of Gal10p for other Gal proteins vary in different organisms. Sequence and structure comparison of Epimerase enzyme showed that the orthologs in K.lactis and S.cervisiae are more similar to each other as compared to the ortholog in E.coli .The studies carried by us will help in better understanding of the galactose metabolism. The above studies may be applied to Human Gal10p, where it can help in gaining useful insight into Galactosemia disease.     

Cloning and expression analysis of a UDP-galactose/glucose pyrophosphorylase from melon fruit provides evidence for the major metabolic pathway of galactose metabolism in raffinose oligosaccharide metabolizing plants

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by alpha-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.     

Structure-based functional annotation: yeast ymr099c codes for a D-hexose-6-phosphate mutarotase

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 alpha and beta 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.     

Structural analysis of the Y299C mutant of Escherichia coli UDP-galactose 4-epimerase. Teaching an old dog new tricks

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-Gal and UDP-Glc during normal galactose metabolism. The mammalian form of the enzyme, unlike its Escherichia coli counterpart, can also interconvert UDP-GalNAc and UDP-GlcNAc. One key feature of the epimerase reaction mechanism is the rotation of a 4-ketopyranose intermediate in the active site. By comparing the high resolution x-ray structures of both the bacterial and human forms of the enzyme, it was previously postulated that the additional activity in the human epimerase was due to replacement of the structural equivalent of Tyr-299 in the E. coli enzyme with a cysteine residue, thereby leading to a larger active site volume. To test this hypothesis, the Y299C mutant form of the E. coli enzyme was prepared and its three-dimensional structure solved as described here. Additionally, the Y299C mutant protein was assayed for activity against both UDP-Gal and UDP-GalNAc. These studies have revealed that, indeed, this simple mutation did confer UDP-GalNAc/UDP-GlcNAc converting activity to the bacterial enzyme with minimal changes in its three-dimensional structure. Specifically, although the Y299C mutation in the bacterial enzyme resulted in a loss of epimerase activity with regard to UDP-Gal by almost 5-fold, it resulted in a gain of activity against UDP-GalNAc by more than 230-fold.     

The role of UDP‐glucose epimerase in carbohydrate metabolism of Arabidopsis

Uridine 5′-diphospho-glucose-4-epimerase (UDP-Glc epimerase) catalyses the reversible epimerization of UDP-galactose and UDP-glucose. In contrast to bacteria and yeast, expression of the UDP-Glc epimerase gene in Arabidopsis was found not to be induced by galactose. To elucidate the metabolic role of this enzyme, transgenic Arabidopsis plants expressing the respective cDNA in sense or antisense orientation were constructed, leading to a range of plant lines with different UDP-Glc epimerase activities. No alterations in morphology were observed and the relative amounts of different galactose-containing compounds were not affected if the plants were raised on soil. However, on agar plates in the presence of galactose, the growth of different lines was increasingly repressed with decreasing enzyme activity, and an increase in the UDP-Gal content was observed in parallel, whereas the UDP-Glc content was nearly constant. The amount of galactose in the cell wall was increased in plants with low UDP-Glc epimerase activity grown on galactose, whereas the cellulose content in the leaves was not altered. Furthermore, starch determined at different times of the day was highly abundant in plants with low UDP-Glc epimerase activity in the presence of galactose. It is proposed that low endogenous UDP-Glc epimerase activity is responsible for the galactose toxicity of the wild-type. Possible mechanisms by which the starch content might be modulated are discussed.     

The regulatory roles of the galactose permease and kinase in the induction response of the GAL network in Saccharomyces cerevisiae

The GAL genetic switch of Saccharomyces cerevisiae exhibits an ultrasensitive response to the inducer galactose as well as the "all-or-none" behavior characteristic of many eukaryotic regulatory networks. We have constructed a strain that allows intermediate levels of gene expression from a tunable GAL1 promoter at both the population and the single cell level by altering the regulation of the galactose permease Gal2p. Similar modifications to other feedback loops regulating the Gal80p repressor and the Gal3p signaling protein did not result in similarly tuned responses, indicating that the level of inducer transport is unique in its ability to control the switch response of the network. In addition, removal of the Gal1p galactokinase from the network resulted in a regimed response due to the dual role of this enzyme in galactose catabolism and transport. These two activities have competing effects on the response of the network to galactose such that the transport effects of Gal1p are dominant at low galactose concentrations, whereas its catabolic effects are dominant at high galactose concentrations. In addition, flow cytometry analysis revealed the unexpected phenomenon of multiple populations in the gal1delta strains, which were not present in the isogenic GAL1 background. This result indicates that Gal1p may play a previously undescribed role in the stability of the GAL network response.     

High resolution X-ray structure of galactose mutarotase from Lactococcus lactis

Galactose mutarotase plays a key role in normal galactose metabolism by catalyzing the interconversion of beta-D-galactose and alpha-D-galactose. Here we describe the three-dimensional architecture of galactose mutarotase from Lactococcus lactis determined to 1.9-A resolution. Each subunit of the dimeric enzyme displays a distinctive beta-sandwich motif. This tertiary structural element was first identified in beta-galactosidase and subsequently observed in copper amine oxidase, hyaluronate lyase, chondroitinase, and maltose phosphorylase. Two cis-peptides are found in each subunit, namely Pro(67) and Lys(136). The active site is positioned in a rather open cleft, and the electron density corresponding to the bound galactose unequivocally demonstrates that both anomers of the substrate are present in the crystalline enzyme. Those residues responsible for anchoring the sugar to the protein include Arg(71), His(96), His(170), Asp(243), and Glu(304). Both His(96) and His(170) are strictly conserved among mutarotase amino acid sequences determined thus far. The imidazole nitrogens of these residues are located within hydrogen bonding distance to the C-5 oxygen of galactose. Strikingly, the carboxylate group of Glu(304) is situated at approximately 2.7 A from the 1'-hydroxyl group of galactose, thereby suggesting its possible role as a general acid/base group.     

Human UDP-galactose 4-epimerase. Accommodation of UDP-N-acetylglucosamine within the active site

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose during normal galactose metabolism. One of the key structural features in the proposed reaction mechanism for the enzyme is the rotation of a 4'-ketopyranose intermediate within the active site pocket. Recently, the three-dimensional structure of the human enzyme with bound NADH and UDP-glucose was determined. Unlike that observed for the protein isolated from Escherichia coli, the human enzyme can also turn over UDP-GlcNAc to UDP-GalNAc and vice versa. Here we describe the three-dimensional structure of human epimerase complexed with NADH and UDP-GlcNAc. To accommodate the additional N-acetyl group at the C2 position of the sugar, the side chain of Asn-207 rotates toward the interior of the protein and interacts with Glu-199. Strikingly, in the human enzyme, the structural equivalent of Tyr-299 in the E. coli protein is replaced with a cysteine residue (Cys-307) and the active site volume for the human protein is calculated to be approximately 15% larger than that observed for the bacterial epimerase. This combination of a larger active site cavity and amino acid residue replacement most likely accounts for the inability of the E. coli enzyme to interconvert UDP-GlcNAc and UDP-GalNAc.     

High resolution x-ray structure of tyvelose epimerase from Salmonella typhi

Tyvelose epimerase catalyzes the last step in the biosynthesis of tyvelose by converting CDP-d-paratose to CDP-d-tyvelose. This unusual 3,6-dideoxyhexose occurs in the O-antigens of some types of Gram-negative bacteria. Here we describe the cloning, protein purification, and high-resolution x-ray crystallographic analysis of tyvelose epimerase from Salmonella typhi complexed with CDP. The enzyme from S. typhi is a homotetramer with each subunit containing 339 amino acid residues and a tightly bound NAD+ cofactor. The quaternary structure of the enzyme displays 222 symmetry and can be aptly described as a dimer of dimers. Each subunit folds into two distinct lobes: the N-terminal motif responsible for NAD+ binding and the C-terminal region that harbors the binding site for CDP. The analysis described here demonstrates that tyvelose epimerase belongs to the short-chain dehydrogenase/reductase superfamily of enzymes. Indeed, its active site is reminiscent to that observed for UDP-galactose 4-epimerase, an enzyme that plays a key role in galactose metabolism. Unlike UDP-galactose 4-epimerase where the conversion of configuration occurs about C-4 of the UDP-glucose or UDP-galactose substrates, in the reaction catalyzed by tyvelose epimerase, the inversion of stereochemistry occurs at C-2. On the basis of the observed binding mode for CDP, it is possible to predict the manner in which the substrate, CDP-paratose, and the product, CDP-tyvelose, might be accommodated within the active site of tyvelose epimerase.     

Microbial β-Galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction

Hydrolysis efficiency of β-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial β-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnβGal), Aspergillus oryzae (AoβGal), Bacillus circulans (BcβGal), Bifidobacterium bifidum (BbβGal), and Kluyveromyces lactis (KlβGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnβGal), Asn140Ala and Asn199Ala (AoβGal), Arg111Ala and Glu355Ala (BcβGal), Arg122Ala and Phe358Ala (BbβGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlβGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcβGal, BbβGal, and KlβGal. The double mutations in BcβGal and BbβGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlβGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of β-galactosidases and rendering them promising for industrial applications.