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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bacteriophage SF6 antigenically converts Shigella flexneri serotype Y strains (-;3,4) to type 3b carrying group antigen 6,3,4 by means of an O-acetylation of the O-antigenic polysaccharide chain. The gene for O-acetyl transferase of bacteriophage SF6 has been cloned, identified and sequenced. The predicted O-acetyl transferase protein encoded by this gene was found to consist of 333 amino acids, (37,185 daltons) and to have some similarity with the galactose-1-phosphate uridylyltransferase protein of Escherichia coli. The gene has been shown to function in a live vaccine strain of S. flexneri Y type (delta aroD), making it a 3b type. The converted type 3b strain, SFL1104, was found to elicit significant protection against challenge by both wild-type serotypes 3b and Y in a guinea-pig keratoconjunctivitis model.
Mol Microbiol 1991 Jan
PMID:Molecular characterization of the O-acetyl transferase gene of converting bacteriophage SF6 that adds group antigen 6 to Shigella flexneri. 201 5

Two UTP-utilizing uridylyltransferases which react with both glucose 1-phosphate and galactose 1-phosphate were isolated from cell-free extracts of Entamoeba histolytica. The more specific of these enzymes, glucose-1-phosphate uridylyltransferase, acts preferentially on glucose 1-phosphate, having a maximum velocity 20-fold greater with this substrate than with galactose 1-phosphate. It was purified 200 fold with a 25% yield and has a molecular weight of 45 000. This enzyme requires a reducing agent for stability. The less specific transferase reacts with both hexose phosphates, having a maximum velocity of 1.35 times greater with galactose 1-phosphate. It was purified 1000 fold with a 20% yield, and has a molecular weight of 40 000. The common Leloir enzyme, UDP glucose-hexose-1-phosphate uridylytransferase (EC 2.7.7.12), was not found in this organism. To avoid confusion with the Leloir enzyme our experience suggests that the less specific enzyme, which is presently referred to in the literature as galactose-1-phosphate uridylyltransferase (EC 2.7.7.10), should be named UTP:hexose-1-phosphate uridylyltransferase (EC 2.7.7.?). The more specific enzyme (EC 2.7.7.9) should be more clearly named UTP:glucose-1-phosphate uridylyltransferase.
Mol Biochem Parasitol 1983 Feb
PMID:Separation and characterization of two UTP-utilizing hexose phosphate uridylyltransferases from Entamoeba histolytica. 630 12

Mutants of Kluyveromyces lactis with elevated uninduced levels of beta-galactosidase (EC 32.1.2.3) activity, constitutive mutants (lac10c), were isolated and characterized to determine the basis for their constitutiveness. These lesions are not operator-type regulatory mutants because they are not closely linked to the beta-galactosidase structural gene. In a constitutive strain having a 7-fold increase in beta-galactosidase activity, the concentration of beta-galactosidase messenger ribonucleic acid (mRNA) was 8- to 10-fold higher than uninduced wild type. The half-life of beta-galactosidase mRNA was the same in the mutant strain (t1/2 = 4.5 +/- 0.2 min) as in uninduced wild-type cells (t1/2 = 4.8 +/- 0.1 min), indicating that the elevated mRNA level in the mutant was not due to a decreased rate of mRNA degradation. Consequently, we hypothesize that the LAC10 product regulates transcription of the beta-galactosidase gene; it probably affects the rate of transcription initiation. Parallel increases in enzyme protein, in constitutive levels of beta-galactosidase activity, and in mRNA further support this position, making translational or posttranslational control by LAC10 unlikely. Several types of data suggest that the LAC10 product functions as a negative regulatory element to prevent transcription. Other data demonstrate that lac10c mutations have pleiotrophic effects, there being constitutive levels not only of beta-galactosidase activity, but also the other lactose-inducible activities of galactokinase (EC 2.7.5.1), galactose-1-phosphate uridyl transferase (EC 2.7.7.10), and lactose transport. It would appear that LAC10 regulates lactose-inducible proteins.
Mol Cell Biol 1981 Nov
PMID:Genetic regulation: yeast mutants constitutive for beta-galactosidase activity have an increased level of beta-galactosidase messenger ribonucleic acid. 681 93

Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT.
Biochem Mol Med 1995 Dec
PMID:Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system. 882 75

Galactosemia is a clinically heterogeneous autosomal recessive inborn error of metabolism caused by deficiency of galactose-1-phosphate uridylyltransferase (GALT). Despite the numerous point mutations identified in the GALT gene, the prevalence of these mutations in different ethnic groups has not been studied. Reports on genotype/phenotype correlation are not consistent due to the small sample sizes studied and the lack of a sensitive enzyme assay. We applied multiplex PCR/ASO dot blot analysis to screen 293 galactosemic patients for 17 known point mutations in exons 5, 6, and 10. Our data demonstrate that only 7 of these mutations were detected in our patients, accounting for 65% of the GALT mutant alleles. Although Q188R is the most common mutation in Caucasian and Hispanic patients, the S135L mutation is most common in African-Americans. Another mutation, F171S, was observed only among African-American patients. An improved, sensitive, and accurate method was used to measure GALT activity in patient's red blood cells. The results indicated that patients homozygous for Q188R have no enzyme activity while those homozygous for S135L had residual enzyme activity. Interestingly, both Q188R/S135L and S135L/F171S compound heterozygotes demonstrated zero enzyme activity. Overall, 85% of Q188R compound heterozygotes also did not have any enzyme activity, whereas the remaining Q188R and the majority of S135L compound heterozygotes expressed variable amounts of GALT activity. We speculate that heterodimeric subunit interaction plays an important role in determining the overall enzymatic activity. Various genotypes thus result in biochemical and clinical heterogeneity among the patients.
Mol Genet Metab 1998 Apr
PMID:Molecular and biochemical basis of galactosemia. 963 94

The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.
Mol Genet Metab 2000 Aug
PMID:Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae. 1099 14

We previously identified a missense mutation at amino acid 135 of human galactose 1-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was substituted for a serine (TCG), S135L. This mutation was common in black patients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts. However, there was residual GALT activity and protein in their leukocytes, and they had near normal total body [13C]galactose oxidation to 13CO2 in breath. To evaluate the biochemical mechanism(s) producing these effects, we overexpressed hGALT proteins with site-directed mutations in this nonconserved amino acid in a GALT-minus Escherichia coli. Enzyme activities detected in bacterial lysates overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H135, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respectively. Only the threonine substitution (S135T) had significant enzyme activity in these lysates. There was also decreased abundance of all mutant proteins in the lysates exposed to bacterial proteolysis during preparation and analysis. This added the variable of bio-instability to analysis of enzyme activities in lysates. To further characterize the catalytic role of serine at amino acid 135 and to differentiate bio-instability from impaired catalysis by the leucine substitution, we purified wild-type and L135-hGALT proteins to homogeneity and analyzed identical amounts of enzyme protein. We found that the apparent Vmax of the purified L135-hGALT protein was significantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 micromol glucose 1-phosphate released/min/mg hGALT protein with no increase in KM for galactose 1-phosphate for the second displacement. The first displacement reaction, although three orders of magnitude slower, was similar between the wild type and L135-hGALT. We conclude that a hydroxyl group on amino acid 135 is required for the catalysis of uridyl transfer from UDP-glucose to UDP-galactose in the presence of galactose 1-phosphate, and plays a role in the bio-stability of hGALT.
Mol Genet Metab
PMID:Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia. 1159 23

Lactose is at present the only soluble carbon source which can be used economically for the production by Hypocrea jecorina (= Trichoderma reesei) of cellulases or heterologous proteins under the control of cellulase expression signals. However, the mechanism by which lactose triggers the formation of cellulases is unknown. To enhance our understanding of lactose metabolism and its relationship to cellulase formation, we have cloned and characterized the gal7 gene (for galactose-1-phosphate uridylyltransferase) of H. jecorina. The gene encodes a polypeptide of 43.8 kDa, the sequence of which exhibits a moderate level of identity (about 50%) to that of the Gal7 proteins of Saccharomyces cerevisiae and Kluyveromyces lactis, and contains an active-site signature typical for galactose-1-phosphate uridylyltransferase family 1. H. jecorina gal7 is not clustered with other genes of galactose metabolism. A single 1.7-kb transcript is synthesized constitutively during the rapid growth phase and accumulated to twice this level during incubation in the presence of D-galactose and L-arabinose and the corresponding polyols (dulcitol, arabitol). A gal7 deletion mutant, constructed by replacing the gal7 reading frame by the H. jecorina pyr4 gene, was unable to grow on D-galactose between pH 4.5 and 7.5, thus proving that in H. jecorina gal7 is essential for metabolism of D-galactose, whereas the growth rate of the mutant on lactose was only reduced by about 50%. The rate of formation of cellobiohydrolase Cel7A and the abundance of the corresponding (cbh1) transcript during growth on lactose was only slightly lower in the absence of gal7, but a significant delay in decay of the cbh1 transcript was noted during later stages of growth. The results suggest that H. jecorina uses only the Leloir pathway for metabolism of D-galactose and lactose. Furthermore, we conclude that metabolism of lactose past the galactose-1-phosphate step is not essential for cellulase formation.
Mol Genet Genomics 2002 Mar
PMID:Lactose metabolism and cellulase production in Hypocrea jecorina: the gal7 gene, encoding galactose-1-phosphate uridylyltransferase, is essential for growth on galactose but not for cellulase induction. 1191 23

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.
Mol Microbiol 2004 Feb
PMID:The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on d-galactose. 1476 77

Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5' proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5' deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT-PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5' deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms.
Hum Mol Genet 2009 May 01
PMID:Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase. 1922 51


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