UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.     

Immobilization of UDP-galactose 4-epimerase from Escherichia coli on the yeast cell surface

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.     

Cloning, expression and characterization of a UDP-galactose 4-epimerase from Escherichia coli

The gene galE encoding UDP-galactose 4-epimerase was cloned into E. coli BL21(DE3) from the chromosomal DNA of E. coli strain K-12. High expression of the soluble recombinant epimerase was achieved in the cell lysate. In order to evaluate the use of this epimerase in enzymatic synthesis of important -Gal epitopes (oligosaccharides with a terminal Gal1,3Gal sequence), a new radioactivity assay (1,3-galactosyltransferase coupled assay) was established to characterize its activity in producing UDP-galactose from UDP-glucose. Approximately 2700 units (100 mg) enzyme with a specific activity of 27 U mg^–1 protein could be obtained from one liter of bacterial culture. The epimerase was active in a wide pH range with an optimum at pH 7.0. This expression system established a viable route to the enzymatic production of -Gal oligosaccharides to support xenotransplantation research.     

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

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 A. 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 A, b = 83.1 A, c = 132.1 A, and one dimer per asymmetric unit. An interpretable electron density map calculated to 2.5 A 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 beta-pleated sheet flanked on either side of alpha-helices. The seventh strand of the beta-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 beta-pleated sheet, two major alpha-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.     

UDP-galactose 4′-epimerase from vicia faba seeds

UDP-Galactose 4′-epimerase was purified ca 800-fold through a multi-step procedure which included affinity chromatography using NAD⁺ -Agarose. Three forms of the enzyme were separated by gel-filtration but only the major form was purified. The pH optimum of the enzyme was 9.5. Exogenous NAD⁺ was not required for enzymic activity but its removal caused inactivation. The enzyme was unstable below pH 7.0 but stable at pH 8.0 in the presence of glycerol and at ?20° for two months. The equilibrium constant for the enzyme-catalysed reaction was 3.2 ± 0.15. The K_m for UDP-galactose and UDP-glucose were 0.12 mM and 0.25 mM, respectively. The inhibition by NADH was competitive, with a K_i of 5 μM. The MW of the enzyme was 78 000; the two minor forms showed the values of 158 000 and 39 000, respectively.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: existence of subunit independent functional site

UDP-galactose 4-epimerase from Kluyveromyces fragilis is a stable homodimer of 75 kDa/subunit with non-covalently bound NAD acting as cofactor. Partial proteolysis with trypsin in the presence of 5'-UMP, a strong competitive inhibitor, led to a degraded product which was purified. Results from SDS-PAGE, size-exclusion (SE)-HPLC and ultracentrifugation indicated its monomeric status and size between 43 and 45 kDa. 'Two-step assay' with UDP-glucose dehydrogenase as coupling enzyme in the presence of NAD ensured epimerase activity of the monomer. The possibility of transient dimerization of monomeric epimerase during catalysis was excluded by SE-HPLC in the presence of excess substrate and NAD. This truncated enzyme retained catalytic site related properties like Km for UDP-galactose, 'NADH-like coenzyme fluorescence' and 'reductive inhibition' similar to its dimeric counterpart. Reversible reactivation of the monomer was achieved up to 95% within 3 min from 8 M urea induced unfolded state, indicating that the catalytic site could form independent of its quaternary structure. Equilibrium unfolding between 0 and 8 M urea indicated that the monomer was less stable compared to the dimer. Chemical modification of amino acids and reconstitution with etheno-NAD suggested that the architecture around the catalytic site of the monomer was conserved. Specific modification reagents further confirmed that the cysteine residues required for catalysis and coenzyme fluorophore reside exclusively on a single subunit negating a 'subunit sharing model' of its catalytic site.     

Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila

Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6xHis-rGalE was determined to be 38271.477 (m/z). The 6xHis-rGalE with a K(m) of 0.5 mM for UDP-galactose exhibited optimum activity at 37 degrees C and pH 8-9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.     

Crystal structure of UDP-galactose 4-epimerase from the hyperthermophilic archaeon Pyrobaculum calidifontis

The crystal structure of a highly thermostable UDP-galactose 4-epimerase (GalE) from the hyperthermophilic archaeon Pyrobaculum calidifontis was determined at a resolution of 1.8 Å. The asymmetric unit contained one subunit, and the functional dimer was generated by a crystallographic two-fold axis. Each monomer consisted of a Rossmann-fold domain with NAD bound and a carboxyl terminal domain. The overall structure of P. calidifontis GalE showed significant similarity to the structures of the GalEs from Escherichia coli, human and Trypanosoma brucei. However, the sizes of several surface loops were markedly smaller in P. calidifontis GalE than the corresponding loops in the other enzymes. Structural comparison revealed that the presence of an extensive hydrophobic interaction at the subunit interface is likely the main factor contributing to the hyperthermostability of the P. calidifontis enzyme. Within the NAD-binding site of P. calidifontis GalE, a loop (NAD-binding loop) tightly holds the adenine ribose moiety of NAD. Moreover, a deletion mutant lacking this loop bound NAD in a loose, reversible manner. Thus the presence of the NAD-binding loop in GalE is largely responsible for preventing the release of the cofactor from the holoenzyme.     

Purification and preliminary X-ray crystallographic studies of recombinant L-ribulose-5-phosphate 4-epimerase from Escherichia coli.

The araD gene from Escherichia coli, coding for L-ribulose-5-phosphate 4-epimerase, was overexpressed and the resulting enzyme was purified to homogeneity. Crystals of L-ribulose-5-phosphate 4-epimerase, obtained with 4.0 M sodium formate as precipitant, belong to space group P4212 with unit cell dimensions a = b = 107.8 A and c = 281.4 A and diffract to at least 2.2 A resolution. Density measurements of these crystals are consistent with eight subunits in the asymmetric unit.     

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.     

UDP-galactose 4-epimerase from Kluyveromyces fragilis: analysis of its hysteretic behavior during catalysis

UDP-galactose 4-epimerase serves as a prototype model of class II oxidoreductases that use bound NAD as a cofactor. This enzyme from Kluyveromyces fragilis is a homodimer with a molecular mass of 75 kDa/subunit. Continuous monitoring of the conversion of UDP-galactose (UDP-gal) to UDP-glucose (UDP-glu) by the epimerase in the presence of the coupling enzyme UDP-glucose dehydrogenase and NAD shows a kinetic lag of up to 80 s before a steady state is reached. The disappearance of the lag follows first-order kinetics (k = 3.22 x 10(-2) s(-1)) at 25 degrees C at enzyme and substrate concentrations of 1.0 nM and 1 mM, respectively. The observed lag is not due to factors such as insufficient activity of the coupling enzyme, association or dissociation or incomplete recruitment of NAD by epimerase, product activation, etc., but was a true expression of the activity of the prepared enzyme. Dissociation of the bound ligand(s) by heat followed by analysis with reverse-phase HPLC, TLC, UV-absorption spectrometry, mass spectrometry, and NMR showed that in addition to 1.78 mol of NAD/dimer, the epimerase also contains 0.77 mol of 5'-UMP/dimer. The latter is a strong competitive inhibitor. Preincubation of the epimerase with the substrate UDP-gal or UDP-glu replaces the inhibitor and also abolishes the lag, which reappeared after the enzyme was treated with 5'-UMP. The lag was not observed as long as the cells were in the growing phase and galactose in the growth medium was limiting, suggesting that association with 5'-UMP is a late log-phase phenomenon. The stoichiometry and conserved amino acid sequence around the NAD binding site of multimeric class I (classical dehydrogenases) and class II oxidoreductases, as reported in the literature, have been compared. It shows that each subunit is independently capable of being associated with one molecule of NAD, suggestive of two NAD binding sites of epimerase per dimer.     

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.     

Functional analysis of disease-causing mutations in human UDP-galactose 4-epimerase

UDP-galactose 4-epimerase (GALE, EC 5.1.3.2) catalyses the interconversion of UDP-glucose and UDP-galactose. Point mutations in this enzyme are associated with the genetic disease, type III galactosemia, which exists in two forms - a milder, or peripheral, form and a more severe, or generalized, form. Recombinant wild-type GALE, and nine disease-causing mutations, have all been expressed in, and purified from, Escherichia coli in soluble, active forms. Two of the mutations (N34S and G319E) display essentially wild-type kinetics. The remainder (G90E, V94M, D103G, L183P, K257R, L313M and R335H) are all impaired in turnover number (k cat) and specificity constant (k cat/Km), with G90E and V94M (which is associated with the generalized form of galactosemia) being the most affected. None of the mutations results in a greater than threefold change in the Michaelis constant (Km). Protein-protein crosslinking suggests that none of the mutants are impaired in homodimer formation. The L183P mutation suffers from severe proteolytic degradation during expression and purification. N34S, G90E and D103G all show increased susceptibility to digestion in limited proteolysis experiments. Therefore, it is suggested that reduced catalytic efficiency and increased proteolytic susceptibility of GALE are causative factors in type III galactosemia. Furthermore, there is an approximate correlation between the severity of these defects in the protein structure and function, and the symptoms observed in patients.     

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.     

Localization of translation initiation in the message for E. coli UDP-galactose 4-epimerase: the amino terminal sequence

The amino acid sequence of $\text{E.}$$\text{coli}$ UDP-galactose 4-epimerase has been determined through the amino-terminal 28-amino acid residues using an automated protein sequenator. Alignment of UDP-galactose operon messenger RNA and the amino acid sequence of epimerase demonstrates that the first 26 bases in the mRNA are transcribed but do not take part in translation of epimerase.     

High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol.

UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-glucose and UDP-galactose. In recent years, the enzyme has been the subject of intensive investigation due in part to its ability to facilitate nonstereospecific hydride transfer between beta-NADH and a 4-keto hexopyranose intermediate. The first molecular model of the epimerase from E. coli was solved to 2.5 A resolution with crystals grown in the presence of a substrate analogue, UDP-phenol (Bauer AJ, Rayment I, Frey PA, Holden HM, 1992, Proteins Struct Funct Genet 12:372-381). There were concerns at the time that the inhibitor did not adequately mimic the sugar moiety of a true substrate. Here we describe the high-resolution X-ray crystal structure of the ternary complex of UDP-galactose 4-epimerase with NADH and UDP-phenol. The model was refined to 1.8 A resolution with a final overall R-factor of 18.6%. This high-resolution structural analysis demonstrates that the original concerns were unfounded and that, in fact, UDP-phenol and UDP-glucose bind similarly. The carboxamide groups of the dinucleotides, in both subunits, are displaced significantly from the planes of the nicotinamide rings by hydrogen bonding interactions with Ser 124 and Tyr 149. UDP-galactose 4-epimerase belongs to a family of enzymes known as the short-chain dehydrogenases, which contain a characteristic Tyr-Lys couple thought to be important for catalysis. The epimerase/NADH/UDP-phenol model presented here represents a well-defined ternary complex for this family of proteins and, as such, provides important information regarding the possible role of the Tyr-Lys couple in the reaction mechanism.     

Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7

The O-antigen of lipopolysaccharide in Gram-negative bacteria plays an important role in bacterium-host interactions. Escherichia coli O86:B7 O-unit contains five sugar residues: one fucose (Fuc) and two each of N-acetylgalactosamine (GalNAc) and galactose (Gal). The entire O-antigen gene cluster was previously sequenced: orf1 was assigned the gne gene for the biosynthesis of UDP-GalNAc. To confirm this annotation, overexpression, purification, and biochemical characterization of Gne were performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal almost equally well. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc are 370, 295, 323, and 373 microM, respectively. The comparison of kinetic parameters of Gne from Escherichia coli O86:B7 to those of other characterized UDP-GlcNAc/Glc 4-epimerases indicated that it has relaxed specificity toward the four substrates, the first characterized enzyme to have this activity in the O-antigen biosynthesis. Moreover, the calculated kcat/Km values for UDP-GalNAc and UDP-Gal are approximately 2-4 times higher than those for UDP-GlcNAc and UDP-Glc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation (S306Y) resulted in a loss of epimerase activity for non-acetylated substrates by about 5-fold but totally abolished the activity for N-acetylated substrates, indicating that residue S306 plays an important role in the determination of substrate specificity.