The human galactose-1-phosphate uridyltransferase gene.

Classical galactosemia is an inborn error of metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (GALT). Standard treatment with dietary galactose restriction will reverse the potentially lethal symptoms of the disease that are manifest in the newborn period. However, the long-term prognosis for these patients is variable. As a first step toward investigating the molecular basis for phenotypic variation in galactosemia, we have cloned and sequenced the entire gene for human galactose-1-phosphate uridyltransferase. This gene is organized into 11 exons spanning 4 kb. In exons 6, 9, and a portion of 10, there is a high degree of amino acid sequence conservation among Escherichia coli, yeast, mouse, and human. We have identified a number of nucleotide changes in the GALT genes of galactosemic patients that alter conserved amino acids. The most common of these is an A to G transition at nucleotide position 1470, converting a glutamine to an arginine at amino acid codon position 188 (Q188R).(ABSTRACT TRUNCATED AT 250 WORDS)     

The biochemical role of glutamine 188 in human galactose-1-phosphate uridyltransferase

The substitution of arginine for glutamine at amino acid 188 (Q188R) ablates the function of human galactose-1-phosphate uridyltransferase (GALT) and is the most common mutation causing galactosemia in the white population. GALT catalyzes two consecutive reactions. The first reaction binds UDP-glucose (UDP-Glu), displaces glucose-1-phosphate (glu-1-P), and forms the UMP-GALT intermediate. In the second reaction, galactose-1-phosphate (gal-1-P) is bound, UDP-galactose (UDP-Gal) is released, and the free enzyme is recycled. In this study, we modeled glutamine, asparagine, and a common mutation arginine at amino acid 188 on the three-dimensional model of the Escherichia coli GALT-UMP protein crystal. We found that the amide group of the glutamine side chain could provide two hydrogen bonds to the phosphoryl oxygens of UMP with lengths of 2.52 and 2.82 A. Arginine and asparagine could provide only one hydrogen bond of 2. 52 and 3.02 A, respectively. To test this model, we purified recombinant human Gln188-, Arg188-, and Asn188-GALT and analyzed the first reaction in the absence of gal-1-P by quantitating glu-1-P released using enzyme-linked methods. Gln188-GALT displaced 80 +/- 7. 0 nmol glu-1-P/mg GALT/min in the first reaction. By contrast, both Arg188- and Asn188-GALT released more glu-1-P (170 +/- 8.0 and 129 +/- 28.4 nmol/mg GALT/min, respectively). The overall, double displacement reaction was quantitated in the presence of gal-1-P. Gln188-GALT produced 80,030 +/- 5,910 nmol glu-1-P/mg GALT/min, whereas the mutant Arg188- and Asn188-GALT released only 600 +/- 71. 2 and 2960 +/- 283.6 nmole glu-1-P/mg GALT/min, respectively. We conclude from these data that glutamine at position 188 stabilizes the UMP-GALT intermediate through hydrogen bonding and enables the double displacement of both glu-1-P and UDP-Gal. The substitution of arginine or asparagine at position 188 reduces hydrogen bonding and destabilizes UMP-GALT. The unstable UMP-GALT allows single displacement of glu-1-P with release of free GALT but impairs the subsequent binding of gal-1-P and displacement of UDP-Gal.     

Separation and characteristics of galactose-1-phosphate and glucose-1-phosphate uridyltransferase from fruit peduncles of cucumber

Galactose-1-phosphate uridyltransferase (EC 2.7.7.10), responsible for the conversion of galactose-1-phosphate (Gal-1-P) to uridine diphosphate galactose (UDPgal) was examined in fruit peduncles of Cucumis sativus L. Two uridyltransferases (pyrophosphorylases), from I and II, were partially purified and resolved on a diethylamino-ethyl-cellulose column. Form I can utilize glucose-1-phosphate (Glc-1-P), while form II can utilize either Gal-1-P or Glc-1-P, with a preference for Gal-1-P. Form I was more heat stable than form II. Both Glc-1-P and Gal-1-P activities of form II were inactivated at the same rate by heating. The finding of a uridyltransferase with preference for Gal-1-P indicates that cucumber may have a Gal-1-P uridyltransferase (pyrophosphorylase) pathway for the catabolism of stachyose in the peduncles. The absence of the enzyme UDP-glucose-hexose-1-phosphate uridyltransferase (EC 2.7.7.12) in this tissue rules out catabolism by the classical Leloir pathway. The incorporation of carbon from UDPglc into Glc-1-P as opposed to sucrose may be regulated by the activities of the uridyltransferases. Pyrophosphate, in the same concentration range, inhibits UDP-gal formation (K_i=0.58±0.10 mM) and stimulates Glc-1-P formation. The ratio of units of pyrophosphatase to units of Gal-1-P uridyltransferase was higher in peduncles from growing fruit than from unpollinated fruit. Modulation of carbon partitioning through a uridyltransferase pathway may be a factor controlling growth of the cucumber fruit.Abbreviations Gal-1-P Galactose-1-phosphate - Glc-1-P glucose-1-phosphate - UDPgal uridine diphosphate galactose - UDPglc uridine diphosphate glucose Paper No. 6908 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned     

Characteristics of galactokinase and galactose-1-phosphate uridyltransferase in cultivated fibroblasts and amniotic fluid cells

Summary The kinetic characteristics of galactose-1-phosphate uridyltransferase and galactokinase in cultivated fibroblasts and amniotic fluid cells were investigated. The K _m values of galactokinase for galactose at 2.0 mM ATP are 0.34 mM in amniotic fluid cells and 0.48 mM in fibroblasts. The K _m values for ATP at 0.5 mM galactose are 1.25 mM and 2.10 mM.Transferase and galactokinase activities and protein content increase logarithmically during the growth of cultivated cells. The specific activity of both enzymes also increases and reaches a maximum level 10–15 days after subculture. The specific activity of transferase increases faster than that of galactokinase in the case of amniotic fluid cells. In the case of fibroblasts the specific activity of galactokinase increases faster than that of transferase.     

Factors regulating the specific activity of galactose-1-phosphate uridyltransferase during perfusion of suckling rat liver

The regulation of the specific activity of galactose-1-phosphate uridyltransferase (E.C. 2.7.7.12) has been studied using the hexose-perfused suckling rat liver as a model. Perfusion in the recirculating model with galactose concentrations up to 4mm resulted in enzyme activity which fluctuated significantly during a 90-min experimental period. A concentration of 10 mm galactose stabilized the enzyme activity at levels found in unperfused liver. The same stabilizing effect was seen with 10 mm glucose. The elevated transferase specific activity observed after 30 min recirculating perfusion was not seen when the nonrecirculation mode was employed suggesting a factor in the recirculating medium may mediate this effect.     

Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

Polymorphism of galactose-1-phosphate uridyltransferase in the Albanian and Croatian settlements of Molise (Italy)

Four Albanian and three Croatian communities settled in Molise (Italy) have been investigated for galactose-1-phosphate uridyltransferase (GALT) polymorphism. To obtain a detailed identification of each phenotype, electrophoresis and quantitative enzymatic analysis were performed on all samples. In addition to this isoelectric focusing was utilized to confirm and the D and LA variants. The gene frequencies of the different GALT alleles turned out to be: N = 0.912 (Albanians) and N = 0.868 (Croatians); G = 0.004 (Albanians); D = 0.051 (Albanians) and D = 0.081 (Croatians); LA = 0.033 (Albanians) and LA = 0.051 (Croatians). Both variants show frequencies similar to that observed in other Caucasoid populations.     

Characterization of two missense mutations in human galactose-1-phosphate uridyltransferase: different molecular mechanisms for galactosemia.

We report the molecular characterization of two novel galactosemia mutations that exhibit different molecular phenotypes. Both are of the missense type with low or no residual enzyme activity. The R148W mutation results in an unstable protein, although messenger RNA is still produced. In contrast, the L195P mutation produces stable but inactive immunoreactive protein. The R148W mutation alters an amino acid that is not evolutionarily conserved, while the L195P mutation affects a well-conserved residue nine amino acids down-stream from the putative active site nucleophile. These mutations provide evidence that different mechanisms can result in galactosemia: destabilizing mutations in any given area of the protein and missense mutations in conserved domains of the enzyme resulting in low or no activity. These two mutant alleles represent the fifth and sixth galactosemia mutations and confirm the hypothesis that galactosemia results from a multiplicity of mutations at the molecular level.     

Molecular characterization of two galactosemia mutations and one polymorphism: implications for structure-function analysis of human galactose-1-phosphate uridyltransferase.

We report here the molecular characterization of two galactosemia mutations, L74P and F171S, and one polymorphism, S135L, in human galactose-1-phosphate uridyltransferase (GALT). Both galactosemia mutations result in reduced enzymatic activity when reconstructed in the cDNA and overexpressed. The polymorphism, in contrast, has near normal activity. Both mutations affect evolutionarily conserved residues, suggesting that they are functionally important, while the polymorphism occurs in a nonconserved domain which is presumably not critical for enzymatic function. The F171S mutation is close to the putative active-site nucleophile. Our data further support the notion of molecular heterogeneity of galactosemia and suggest that galactosemia mutations and GALT polymorphisms may be useful tools in highlighting different functional domains in human GALT.     

Characterization of two stop codon mutations in the galactose-1-phosphate uridyltransferase gene of three male galactosemic patients with severe clinical manifestation

Classical galactosemia, which is caused by deficiency of galactose-1-phosphate uridyltransferase, is characterized by acute problems of hepatocellular dysfunction, sepsis, cataracts and failure to thrive. Galactose limitation reverses these symptoms immediately; however, the long-term complications, such as mental retardation and ovarian failures are major problems in most of these patients. In order to investigate the molecular basis for phenotype variation in galactosemia, we have screened the most common mutation in the GALT gene, Q188R. We have further examined those patients who are heterozygous for Q188R or negative for this mutation by SSCP analysis and direct sequencing. In three male patients, we have identified, for the first time, two stop-codon mutations in the GALT gene, G212X (exon 7) and E340X (exon 10). Two patients of 8 and 28 years of age, respectively, who are compound heterozygotes for Q188R and G212X, have severe mental retardation and their general clinical condition is more severe than that of patients with missense mutations. The third patient, who is 8 years of age and who is homozygous for E340X, the N314D polymorphism and a silent substitution L218L, presents with a relatively normal physical and mental condition to date.     

Purification of galactose-1-phosphate uridylyltransferase from human placenta

Galactose-1-phosphate uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been purified 4000-fold from human placenta in four chromatographic steps using DEAE-cellulose, hydrocylapatite, ethyliminohexylagarose, and Sephacryl S-200. The specific activity of the homogeneous enzyme was 56 units/mg protein. The placental enzyme consists of two similar subunits, each of molecular weight about 48,000. The placental enzyme was similar to published results for the red cell enzyme (V. P. Williams, Arch. Biochem. Biophys., 1978, 191, 182–191) with respect to subunit molecular weight, electrophoretic migration, and immunological properties. The more purified fractions of the placental enzyme invariably contained a glycoprotein which was removed in the gel filtration step. After this glycoprotein was removed, the enzyme was very labile and only about 20% of the catalytic activity was recovered.     

A selection system for transgenic plants based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene

A new selection system based on galactose as selective agent and a UDP-glucose:galactose-1-phosphate uridyltransferase gene as selective gene is presented. A broad range of plant species, including agronomically important crops such as maize and rice, is sensitive to low dosages of galactose. The toxicity of galactose is believed to be due to accumulation of galactose-1-phosphate, generated by endogenous galactokinase after uptake. Here, it is demonstrated that this toxicity can be sufficiently alleviated by the Agrobacterium tumefaciens-mediated introduction of the E. coli UDP-glucose:galactose-1-phosphate uridyltransferase (galT) gene, driven by a 35S-promoter, to allow transgenic shoots of potato and oil seed rape to regenerate on galactose containing selection media, resulting in high transformation frequencies (up to 35% for potato). Analysis of genomic DNA and UDP-glucose:galactose-1-phosphate uridyltransferase activity in randomly selected potato transformants confirmed the presence and active expression of the galT gene. The agricultural performance of transgenic potatoes was evaluated by monitoring the phenotype and tuber yield for two generations and these characters were found to be indistinguishable from non-transgenic controls. Thus, the galactose selection system provides a new alternative being distinct from conventional antibiotic and herbicide selection systems as well as so-called positive selection systems where the selective agent has a beneficial effect.     

Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family.

We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent heterogeneity of the human enzyme galactose-1-phosphate uridylyltransferase

Galactose-1-phosphate uridylyltransferase (GALT) acts by a double displacement mechanism, catalyzing the second step in the Leloir pathway of galactose metabolism. Impairment of this enzyme results in the potentially lethal disorder, galactosemia. Although the microheterogeneity of native human GALT has long been recognized, the biochemical basis for this heterogeneity has remained obscure. We have explored the possibility of covalent GALT heterogeneity using denaturing two-dimensional gel electrophoresis and Western blot analysis to fractionate and visualize hemolysate hGALT, as well as the human enzyme expressed in yeast. In both contexts, two predominant GALT species were observed. To define the contribution of uridylylated enzyme intermediate to the two-spot pattern, we exploited the null allele, H186G-hGALT. The Escherichia coli counterpart of this mutant protein (H166G-eGALT) has previously been demonstrated to fold properly, although it cannot form covalent intermediate. Analysis of the H186G-hGALT protein demonstrated a single predominant species, implicating covalent intermediate as the basis for the second spot in the wild-type pattern. In contrast, three naturally occurring mutations, N314D, Q188R, and S135L-hGALT, all demonstrated the two-spot pattern. Together, these data suggest that uridylylated hGALT comprises a significant fraction of the total GALT enzyme pool in normal human cells and that three of the most common patient mutations do not disrupt this distribution.