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Gerard T Berry - One of the best experts on this subject based on the ideXlab platform.
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transient developmental delays in infants with duarte 2 variant galactosemia
Molecular Genetics and Metabolism, 2021Co-Authors: Susan E Waisbren, Cynthia S. Gubbels, Catherine Tran, Didem Demirbas, Margaret Hsiao, Vikram Daesety, Gerard T BerryAbstract:Duarte galactosemia is not classic galactosemia, but rather an example of biochemical variant galactosemia that results in approximately 25% residual activity of Galactose-1-Phosphate Uridylyltransferase (GALT) enzyme. In contrast, classic galactosemia is associated with complete or near complete absence of GALT activity. While infants with classic galactosemia are placed on galactose-restricted diets to prevent the acute and long-term manifestations of their metabolic disorder, while individuals with Duarte variant galactosemia (Duarte-2 galactosemia) do not require diet therapy. The long-term complications that are seen in classic galactosemia such as cerebellar ataxia, and hypergonadotropic hypogonadism do not occur in Duarte-2 galactosemia. While Duarte galactosemia does not appear to be a metabolic disease, it may have an impact on early neurodevelopmental outcomes. This study examined developmental outcomes and the need for special services in individuals with Duarte-2 galactosemia in comparison to individuals with classic galactosemia. We performed a medical record review of individuals with GALT deficiency who were evaluated at Boston Children's Hospital and enrolled in our study of outcomes in galactosemia. This included 95 participants, 21 with Duarte-2 galactosemia and 73 with classic galactosemia. Duarte-2 participants had developmental test scores within the average range. However, 42% of subjects with Duarte-2 galactosemia had participated in early intervention and/or special education and 32% received speech therapy. Their pattern of strengths and weaknesses in cognitive/language/motor domains was similar to that noted in participants with classic galactosemia, albeit to a milder degree. The data indicate that in children with Duarte-2 variant galactosemia, the cognitive/language and motor skills were within normal limits with their cognitive/language skills developing earlier than their motor skills during their first year of life. A history of diet treatment was not related to the use of special services. These results suggest that Duarte-2 galactosemia increases the risk for early mild developmental delays irrespective of treatment history, which resolves over time, and highlights the need to further assess neurodevelopment in early infancy, in Duarte-2 galactosemia. As Duarte-2 galactosemia is not a bona fide biochemical genetic disease, we hypothesize that elements in the genomic space that include the GALT gene are responsible for a transient delay in language-related motor skills during early infancy.
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the ability of an lc ms ms based erythrocyte galt enzyme assay to predict the phenotype in subjects with galt deficiency
Molecular Genetics and Metabolism, 2019Co-Authors: Didem Demirbas, Cynthia S. Gubbels, Minela Haskovic, Xiaoping Huang, Susan E Waisbren, Vikram Daesety, Susan Feenstra, Leah Hecht, Harvey L Levy, Gerard T BerryAbstract:Abstract Background GALT deficiency is a rare genetic disorder of carbohydrate metabolism. Due to the decreased activity or absence of the enzyme Galactose-1-Phosphate Uridylyltransferase (GALT), cells from affected individuals are unable to metabolize galactose normally. Lactose consumption in the newborn period could potentially lead to a lethal disease process with multi-organ involvement. In contrast to the newborn-stage disease, however, a galactose-restricted diet does not prevent long-term complications such as central nervous system (CNS) dysfunction with speech defects, learning disability and neurological disease in addition to hypergonadotropic hypogonadism or primary ovarian insufficiency (POI) in females. As the literature suggests an association between GALT enzyme activity and the long-term complications, it is of importance to have a highly sensitive assay to quantify the GALT enzyme activity. To that end, we had developed a sensitive and accurate LC-MS/MS method to measure GALT enzyme activity. Its ability to predict outcome is the subject of this report. Materials and methods The GALT enzyme activity in erythrocytes from 160 individuals, in which 135 with classic, clinical variant or biochemical variant galactosemia, was quantified by LC-MS/MS. Individuals with GALT deficiency were evaluated for the long-term complications of speech defects, dysarthria, ataxia, dystonia, tremor, POI, as well as intellectual functioning (full scale IQ). The LC-MS/MS results were compared to a variety of assays: radioactive, [14C]-Galactose-1-Phosphate, paper chromatography with scintillation counting, enzyme-coupled assays with spectrophotometric or fluorometric readout or high-pressure liquid chromatography with UV detection of UDP-galactose. Results The LC-MS/MS method measured GALT activity as low as 0.2%, whereas other methods showed no detectable activity. Largely due to GALT activities that were over 1%, the LC-MS/MS measurements were not significantly different than values obtained in other laboratories using other methodologies. Severe long-term complications were less frequently noted in subjects with >1% activity. Patients with a p.Q188R/p.Q188R genotype have no residual enzyme activity in erythrocytes. Conclusion Our LC-MS/MS assay may be necessary to accurately quantify residual GALT activities below 5%. The data suggest that patients with >1% residual activity are less likely to develop diet-independent long-term complications. However, much larger sample sizes are needed to properly assess the clinical phenotype in patients with residual enzyme activities between 0.1 and 5%.
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impaired fertility and motor function in a zebrafish model for classic galactosemia
Journal of Inherited Metabolic Disease, 2018Co-Authors: Jo M Vanoevelen, Gerard T Berry, Ana I Coelho, Rein Vos, Jorgen Bierau, Xiaoping Huang, Britt Van Erven, Estela M RubiogozalboAbstract:Classic galactosemia is a genetic disorder of galactose metabolism, caused by severe deficiency of Galactose-1-Phosphate Uridylyltransferase (GALT) enzyme activity due to mutations of the GALT gene. Its pathogenesis is still not fully elucidated, and a therapy that prevents chronic impairments is lacking. In order to move research forward, there is a high need for a novel animal model, which allows organ studies throughout development and high-throughput screening of pharmacologic compounds. Here, we describe the generation of a galt knockout zebrafish model and present its phenotypical characterization. Using a TALEN approach, a galt knockout line was successfully created. Accordingly, biochemical assays confirm essentially undetectable galt enzyme activity in homozygotes. Analogous to humans, galt knockout fish accumulate Galactose-1-Phosphate upon exposure to exogenous galactose. Furthermore, without prior exposure to exogenous galactose, they exhibit reduced motor activity and impaired fertility (lower egg quantity per mating, higher number of unsuccessful crossings), resembling the human phenotype(s) of neurological sequelae and subfertility. In conclusion, our galt knockout zebrafish model for classic galactosemia mimics the human phenotype(s) at biochemical and clinical levels. Future studies in our model will contribute to improved understanding and management of this disorder.
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galactose 1 phosphate Uridylyltransferase galt gene a novel positive regulator of the pi3k akt signaling pathway in mouse fibroblasts
Biochemical and Biophysical Research Communications, 2016Co-Authors: Bijina Balakrishnan, Anwer Siddiqi, Wyman Chen, Manshu Tang, Xiaoping Huang, Didem Demirbas Cakici, Gerard T BerryAbstract:Abstract The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate Uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan -Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3β (−74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT 2 Profiler PCR Custom Arrays, the mRNA abundance of Akt1 , Pdpk1 , Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.
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N- and O-linked glycosylation of total plasma glycoproteins in galactosemia
Molecular Genetics and Metabolism, 2012Co-Authors: Tyler J. Gleason, Uriel Castañeda, Miao He, Gerard T Berry, Judith L. Fridovich-keilAbstract:Classic galactosemia is a potentially lethal metabolic disorder that results from profound impairment of the enzyme Galactose-1-Phosphate Uridylyltransferase (GALT); despite decades of research, the underlying mechanism of pathophysiology remains unclear. Previous studies of plasma and tissue samples from patients with classic galactosemia have revealed defects of protein and lipid glycosylation, however, the underlying bases for these defects and their clinical significance, if any, has remained unclear. As a step toward addressing these questions we characterized both the N- and O-linked glycomes of plasma proteins from neonates, infants, children, and adults with galactosemia using mass spectrometry and asked (1) whether similar or disparate defects exist for N-linked and O-linked modifications, (2) what factors correlate with the severity of these defects in different patients, and perhaps most important, (3) whether there is any apparent relationship between chronic glycosylation defects and long-term outcome in patients. We found that some but not all of the galactosemic neonates tested exhibited abnormal N- and O-linked glycosylation of plasma proteins. The types of abnormalities seen were similar between N- and O-linked moieties, but the extent of the defects varied between patients. Age, gender, GALT genotype, and predicted residual GALT activity all failed to explain the extent of the glycosylation defect in the samples studied. Dietary galactose restriction markedly normalized both the N- and O-linked glycosylation patterns for all infants tested; however, any remaining glycosylation defects evident in the plasma of older children or adults on galactose-restricted diets showed no correlation with clinical outcome. These data cannot rule out the possibility that subtle or localized glycosylation defects, not detectable by our methods or not reflected in plasma, may contribute to acute or long-term outcome severity.
Ivan Rayment - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of the h166g site directed mutant of galactose 1 phosphate Uridylyltransferase complexed with either udp glucose or udp galactose detailed description of the nucleotide sugar binding site
Biochemistry, 1997Co-Authors: James B Thoden, Ivan Rayment, Frank J Ruzicka, P A Frey, Hazel M HoldenAbstract:Galactose-1-Phosphate Uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P21 with unit cell dimensions of a ) 68 A,b ) 58 A,c ) 189 A, and‚ ) 100° and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the Uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The Uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native Galactose-1-Phosphate Uridylyltransferase.
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the structure of nucleotidylated histidine 166 of galactose 1 phosphate Uridylyltransferase provides insight into phosphoryl group transfer
Biochemistry, 1996Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:Galactose-1-Phosphate Uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049−11061]. In particular, positions of the respective UMP α-phosphoryl groups differ by ∼4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between Ne2 of...
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Structural Analysis of the H166G Site-Directed Mutant of Galactose-1-Phosphate Uridylyltransferase Complexed with either UDP-glucose or UDP-galactose: Detailed Description of the Nucleotide Sugar Binding Site†,‡
1996Co-Authors: James B Thoden, Perry A Frey, Ivan Rayment, Frank J Ruzicka, Hazel M HoldenAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5′-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 Å resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P21 with unit cell dimensions of a) 68 Å, b) 58 Å, c) 189 Å, and â) 100 ° and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the Uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues criticall
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The structure of nucleotidylated histidine-166 of Galactose-1-Phosphate uridylyl transferase provides insight into phosphoryl group transfer. Biochemistry 35:11560–11569
1996Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6 % (data between 65 and 1.86 Å resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP R-phosphoryl groups differ by ∼4 Å. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N2 of the nucleophile and the R-phosphorus of UMP. A hydrogen bond that is conserved in both complexes between His 166 Nδ1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well. Th
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Three-dimensional structure of Galactose-1-Phosphate uridyltransferase from Escherichia coli at 1.8 Å resolution
1995Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDPgalactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multi le isomorphous replacement and electron density modification techniques and has "half-barrel". The barrel staves are formed by nine strands of antiparallel P-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the halfbarrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, Sy of a cysteine is modified by P-mercaptoethanol, which prevents the a-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Led4, Phe75, Am7', Asp78, Phe79, and Va11°8. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations. been refined to 1.8 x resolution. Enzyme subunits consist of a single domain with the topology of a Galactose-1 -phosphate Uridylyltransferase (hexose-1 -phosphate Uridylyltransferase, EC 2.7.7.12) catalyzes the nucleotide exchange between UDP-hexoses and hexose 1-phosphates. This provides an essential balance of UDPglc' and UDP-gal for the cell. Such activated hexose sugars are consumed in the synthesis of disaccharides, glycoproteins, glycolipids, and glycoge
Perry A Frey - One of the best experts on this subject based on the ideXlab platform.
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transient kinetics of formation and reaction of the uridylyl enzyme form of galactose 1 p Uridylyltransferase and its q168r variant insight into the molecular basis of galactosemia
Biochemistry, 1998Co-Authors: Sandaruwan Geeganage, Perry A FreyAbstract:Galactose-1-Phosphate Uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli Uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560−11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type Uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 °C, the first-order rate constant for uridylylation by UDP-glucose is 281 ± 18 s-1, and for deuridylylation it is 226 ± 10 s-1 with Glc-1-P and 166 ± 10 s-1 with Gal-1-P. Inasmuch a...
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the structure of nucleotidylated histidine 166 of galactose 1 phosphate Uridylyltransferase provides insight into phosphoryl group transfer
Biochemistry, 1996Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:Galactose-1-Phosphate Uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049−11061]. In particular, positions of the respective UMP α-phosphoryl groups differ by ∼4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between Ne2 of...
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Structural Analysis of the H166G Site-Directed Mutant of Galactose-1-Phosphate Uridylyltransferase Complexed with either UDP-glucose or UDP-galactose: Detailed Description of the Nucleotide Sugar Binding Site†,‡
1996Co-Authors: James B Thoden, Perry A Frey, Ivan Rayment, Frank J Ruzicka, Hazel M HoldenAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5′-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 Å resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P21 with unit cell dimensions of a) 68 Å, b) 58 Å, c) 189 Å, and â) 100 ° and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the Uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues criticall
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The structure of nucleotidylated histidine-166 of Galactose-1-Phosphate uridylyl transferase provides insight into phosphoryl group transfer. Biochemistry 35:11560–11569
1996Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6 % (data between 65 and 1.86 Å resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J. E., Frey, P. A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP R-phosphoryl groups differ by ∼4 Å. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N2 of the nucleophile and the R-phosphorus of UMP. A hydrogen bond that is conserved in both complexes between His 166 Nδ1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well. Th
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Three-dimensional structure of Galactose-1-Phosphate uridyltransferase from Escherichia coli at 1.8 Å resolution
1995Co-Authors: Joseph E Wedekind, Perry A Frey, Ivan RaymentAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDPgalactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multi le isomorphous replacement and electron density modification techniques and has "half-barrel". The barrel staves are formed by nine strands of antiparallel P-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the halfbarrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, Sy of a cysteine is modified by P-mercaptoethanol, which prevents the a-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Led4, Phe75, Am7', Asp78, Phe79, and Va11°8. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations. been refined to 1.8 x resolution. Enzyme subunits consist of a single domain with the topology of a Galactose-1 -phosphate Uridylyltransferase (hexose-1 -phosphate Uridylyltransferase, EC 2.7.7.12) catalyzes the nucleotide exchange between UDP-hexoses and hexose 1-phosphates. This provides an essential balance of UDPglc' and UDP-gal for the cell. Such activated hexose sugars are consumed in the synthesis of disaccharides, glycoproteins, glycolipids, and glycoge
Hazel M Holden - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of the h166g site directed mutant of galactose 1 phosphate Uridylyltransferase complexed with either udp glucose or udp galactose detailed description of the nucleotide sugar binding site
Biochemistry, 1997Co-Authors: James B Thoden, Ivan Rayment, Frank J Ruzicka, P A Frey, Hazel M HoldenAbstract:Galactose-1-Phosphate Uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P21 with unit cell dimensions of a ) 68 A,b ) 58 A,c ) 189 A, and‚ ) 100° and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the Uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The Uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native Galactose-1-Phosphate Uridylyltransferase.
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Structural Analysis of the H166G Site-Directed Mutant of Galactose-1-Phosphate Uridylyltransferase Complexed with either UDP-glucose or UDP-galactose: Detailed Description of the Nucleotide Sugar Binding Site†,‡
1996Co-Authors: James B Thoden, Perry A Frey, Ivan Rayment, Frank J Ruzicka, Hazel M HoldenAbstract:ABSTRACT: Galactose-1-Phosphate Uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5′-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 Å resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P21 with unit cell dimensions of a) 68 Å, b) 58 Å, c) 189 Å, and â) 100 ° and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the Uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues criticall
Kent Lai - One of the best experts on this subject based on the ideXlab platform.
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novel mrna based therapy reduces toxic galactose metabolites and overcomes galactose sensitivity in a mouse model of classic galactosemia
Molecular Therapy, 2020Co-Authors: Bijina Balakrishnan, Vi Nguyen, Christine Deantonis, Paolo Martini, Kent LaiAbstract:Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate Uridylyltransferase (GALT) gene. Previously, we constructed a GalT−/− (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT−/− newborn pup.
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salubrinal enhances eif2α phosphorylation and improves fertility in a mouse model of classic galactosemia
Biochimica et Biophysica Acta, 2019Co-Authors: Bijina Balakrishnan, Anwer Siddiqi, J Mella, A Lupo, Julie Hollien, Joshua Johnson, Kent LaiAbstract:Abstract Loss of galactose-1 phosphate Uridylyltransferase (GALT) activity in humans results in Classic Galactosemia, and the GalT-deficient (GalT−/−) mouse mimics the patient condition. GalT−/− ovaries display elevated endoplasmic reticulum (ER) stress marker, BiP, and downregulated canonical phosphatidylinositol 3-kinase (Pi3k)/protein kinase B (Akt) growth/pro-survival signaling. Numbers of primordial follicles are reduced in the mutants, recapitulating the accelerated ovarian aging seen in human patients. We previously found that oral administration of the compound Salubrinal (an eIF2α phosphatase inhibitor), resulted in reduction of ovarian BiP expression, rescued Pi3k/Akt signaling, and a doubling of primordial follicles in GalT−/− adults. Here, we further characterized galactosemic stress in GalT−/− mice versus wild-type (WT) controls, and examined whether Salubrinal treatment improved broader reproductive parameters. We assessed the expression levels of factors of the unfolded protein response (UPR), and found that BiP, phospho-Perk, and phospho-eIF2α were all elevated in GalT−/− ovaries. However, neither IKK activation (NFκB pathway) nor alternative Xbp1 splicing downstream of ER membrane protein Ire1α activation was induced, suggesting an Xbp1-independent UPR in galactosemic stress. Moreover, Salubrinal treatment significantly increased the number of ovulated eggs in mutant animals after gonadotrophic superovulation. Salubrinal treatment also normalized estrus cycle stage lengths and resulted in significantly larger litter sizes than vehicle-treated mutants. Overall, we show that Salubrinal protects against galactosemia-induced primordial follicle loss in a fashion that includes suppressing the de-phosphorylation of eIF2α, and that intervention in this way significantly improves and extends ovarian function, fertility, and fecundity.
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reversal of aberrant pi3k akt signaling by salubrinal in a galt deficient mouse model
Biochimica et Biophysica Acta, 2017Co-Authors: B Balakrishnan, Joshua Johnson, C Nicholas, A Siddiqi, Wyman Chen, E Bales, M Feng, Kent LaiAbstract:Classic Galactosemia is an autosomal recessive disorder caused by deleterious mutations in the GALT gene, which encodes galactose-1 phosphate Uridylyltransferase enzyme (GALT: EC 2.7.7.12). Recent studies of primary skin fibroblasts isolated from the GalT-deficient mice demonstrated a slower growth rate, a higher level of endoplasmic reticulum (ER) stress, and down-regulation of the Phosphoinositide 3 kinase/Protein kinase B (PI3K/Akt) signaling pathway. In this study, we compared the expression levels of the PI3K/Akt signaling pathway in normal and GalT-deficient mouse tissues. In mutant mouse ovaries, phospho-Akt [pAkt (Ser473)] and pGsk3β were reduced by 62.5% and 93.5%, respectively (p<0.05 versus normal controls). In mutant cerebella, pAkt (Ser473) and pGsk3β were reduced by 62%, 50%, respectively (p<0.05). To assess the role of ER stress in the down-regulation of PI3K/Akt signaling, we examined if administration of Salubrinal, a chemical compound that alleviates ER stress, to GalT-deficient fibroblasts and animals could normalize the pathway. Our results demonstrated that Salubrinal effectively reversed the down-regulated PI3K/Akt signaling pathway in the mutant cells and animals to levels close to those of their normal counterparts. Moreover, we revealed that Salubrinal can significantly slow down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. These results open the door for a new therapeutic approach for the patients with Classic Galactosemia.
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the leloir pathway of galactose metabolism a novel therapeutic target for hepatocellular carcinoma
Anticancer Research, 2016Co-Authors: Manshu Tang, Enoabasi Etokidem, Kent LaiAbstract:Hepatocellular carcinoma (HCC) is one of the most lethal types of cancer worldwide, with poor prognosis and limited treatments. In order to identify novel therapeutic targets that will lead to development of effective therapies with manageable side effects, we tested the hypothesis that knocking-down galactokinase (GALK1) or galactose-1 phosphate Uridylyltransferase (GALT) gene expression would control the growth of cultured hepatoma cells. Our results showed small interfering RNA (siRNA) against GALK1 or GALT inhibited the growth of HepG2 cells in culture. Western blot analysis revealed simultaneous down-regulation of multiple players of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) growth signaling pathway, as well as heat-shock protein 90 (HSP90) and poly ADP ribose polymerase (PARP). Reverse transcription-polymerase chain reaction (RT-PCR) data, however, showed no significant mRNA reduction of the encoded genes. Our study thus not only supports GALK1 and GALT as being possible novel targets for treating HCC, but also uncovers new post-transcriptional regulatory mechanisms that link the galactose metabolic pathway to protein expression of the PI3K/AKT pathway in hepatoma.
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innovative therapy for classic galactosemia tale of two hts
Molecular Genetics and Metabolism, 2012Co-Authors: Manshu Tang, Sina I Odejinmi, Hariprasad Vankayalapati, Klaas J Wierenga, Kent LaiAbstract:Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of Galactose-1-Phosphate Uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that Galactose-1-Phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts toward identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibits glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies.
