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Classic galactosemia is a rare disease with long-term consequences that seriously affect the quality of life of patients. To date, various therapeutic approaches are being developed, but treatments that target the molecular defects in the mutant galactose-1-phosphate uridylyltransferase ( GALT ) gene are lacking. We conducted a computational search for putative pharmacochaperones by applying a drug repurposing strategy, and we found that one compound, already active as a pharmacochaperone in another pathology, doubled the enzymatic activity of the purified mutant enzyme in an in vitro test. Furthermore, an extensive computational search in a database of known active molecules found another compound able in its turn to improve in vitro enzymatic activity. Both compounds are predicted to interact with a cavity at the enzyme interface previously supposed to be an allosteric site for the GALT enzyme. In vitro tests confirmed also the reduced accumulation of galactose-1-phosphate (G1P) in fibroblasts of patients. Although these results must be considered preliminary, our findings pave the way for future research lines focused on the search for promising pharmacochaperones that can directly rescue the activity of the enzyme.

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg 2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia., Competing Interests: Declaration of competing interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Classic galactosemia (CG) is a rare inborn error of metabolism that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection and rapid and lifelong dietary restriction of galactose, which is the current standard of care, most patients grow to experience a broad range of complications that can include motor difficulties. The goal of this study was to characterize hand fine motor control deficit among children and adults with classic galactosemia (CG). Specifically, we used Neuroglyphics software to collect digital Archimedes spiral drawings on a touch screen from 57 volunteers with CG (cases) and 80 controls. Hand fine motor control was scored as root mean square (RMS) of spirals drawn relative to an idealized template. Presence of tremor was defined as a peak in periodicity of changes in drawing speed or direction in the 4-8 Hz range. We observed a highly significant difference (P < .001) in RMS scores between cases and controls, with almost 51% of cases showing at least 1 of 4 spirals scoring outside the 95th percentile for controls. The corresponding prevalence for controls was 10%. Similarly, more than 35% of cases, and almost 14% of controls, showed at least 1 of 4 spirals with a tremor amplitude above the 95th % cutoff for controls. Our results both confirm and extend what is known about hand fine motor control deficit among children and adults with CG and establish digital assessment as a useful approach to quantify this outcome., (© 2021 SSIEM.)

Classic Galactosaemia is a genetic disorder, characterised by galactose intolerance in newborns. It occurs due to recessive mutations in the galactose-1-phosphate uridylyltransferase (GALT) gene. One of the main alterations caused by GALT deficiency is the accumulation of galactose 1-phosphate (Gal-1P) in cells. Studies have suggested that Gal-1P exerts cellular toxicity, possibly by inhibiting cellular metabolism. However, the exact significance of Gal-1P in disease pathogenesis remains unclear. In this study, we tested the hypothesis that Gal-1P inhibits cellular glucose utilisation by competing with substrates in the glycolytic pathway. We also investigated the metabolism of both galactose and glucose in GALT-expressing HEK293T and 143B cells to identify critical reactions steps contributing to the metabolic toxicity of galactose. Notably, we found that galactose-treated HEK293T and 143B cells, which express endogenous GALT, accumulate markedly high intracellular Gal-1P concentrations. Despite very high intracellular Gal-1P concentrations, no inhibition of cellular glucose uptake and no significant changes in the intracellular concentrations of glycolytic metabolites were observed. This indicates that Gal-1P does not exert an inhibitory effect on glycolysis in cells and rules out one potential hypothesis for cellular Gal-1P toxicity. We also investigated the mechanism responsible for the observed Gal-1P accumulation. Our results suggest that Gal-1P accumulation is a result of both low GALT activity and the absence of product inhibition by Gal-1P on galactokinase (GALK1), the enzyme responsible for phosphorylating galactose to Gal-1P. These findings provide a better understanding of the disease mechanisms underlying Classic Galactoaemia., (© 2019 SSIEM.)

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction., (© 2019 Wiley Periodicals, Inc.)

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism, if untreated, that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme of the Leloir pathway of galactose metabolism. While newborn screening and rapid dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms of CG, by mid-childhood, most treated patients experience significant complications. The mechanisms underlying these long-term deficits remain unclear. Here we introduce a new GALT-null rat model of CG and demonstrate that these rats display cataracts, cognitive, motor, and growth phenotypes reminiscent of patients outcomes. We further apply the GALT-null rats to test how well blood biomarkers, typically followed in patients, reflect metabolic perturbations in other, more relevant tissues. Our results document that the relative levels of galactose metabolites seen in GALT deficiency differ widely by tissue and age, and that red blood cell Gal-1P, the marker most commonly followed in patients, shows no significant association with Gal-1P in other tissues. The work reported here establishes our outbred GALT-null rats as an effective model for at least four complications characteristic of CG, and sets the stage for future studies addressing mechanism and testing the efficacy of novel candidate interventions., (© 2019 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background: The pathogenesis of classical galactosemia, a rare metabolic disorder associated with developmental complications in neonates and children due to inherited deficiency of galactose-1-phosphate (Gal-1-P) uridylyltransferase (GALT), is known to be mediated by elevated Gal-1-P levels and involves a cascade of cytokines, reactive oxygen species (ROS) and growth factors., Objectives: To examine ex vivo the effect of Gal-1-P on the mitogenic activity of different growth factors, particularly insulin-like growth factor-1 (IGF-1), known to regulate growth and development from the fetal stage to adulthood., Material and Methods: Fibroblasts derived from the foreskin of 3-8-day-old healthy neonates were cultured for 1-14 days with 0-20 mM galactose or 0-10 mM Gal-1-P and then stimulated with 5% fetal bovine serum (FBS) or 50 ng/mL of platelet-derived growth factor (PDGF) or fibroblast growth factor (FGF) or IGF-1 for 24 h. DNA synthesis was measured and protein expression of PDGFR, FGFR and IGF-1R was assessed with western blotting., Results: Supra-physiological concentrations of galactose significantly decreased FBSand IGF-1-induced BrdU incorporation. The presence of Gal-1-P (5-10 mM) in culture medium for 7-14 days significantly (p < 0.01) decreased IGF-1-, PDGFand FBS-stimulated DNA synthesis. While treatment with Gal-1-P selectively and significantly (p < 0.01) reduced the protein expression of IGF-1 receptor, galactose treatment did not have any marked effect on examined growth factor receptors., Conclusions: This study demonstrates that Gal-1-P impairs IGF-1 activity through IGF-1-receptor impairment, thereby providing a new insight into the molecular mechanisms of galactosemia pathogenesis.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4 , a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4 Δ phenotype is ERAD-independent. We also observed that the ubx4 Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4 Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia., (© 2020 De-Souza et al.)

Background: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes., Methods: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U 13 C]Gal-1-P/ [ 13 C 6 ]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated., Results: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001)., Conclusions: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Clinical outcome of patients with a classical presentation of galactosemia (classical patients) varies substantially, even between patients with the same genotype. With current biomarkers, it is not possible to predict clinical outcome early in life. The aim of this study was to develop a method to provide more insight into galactose metabolism, which allows quantitative assessment of residual galactose metabolism in galactosemia patients. We therefore developed a method for galactose metabolite profiling (GMP) in fibroblasts using [U- 13 C]-labeled galactose., Methods: GMP analysis was performed in fibroblasts of three classical patients, three variant patients and three healthy controls. The following metabolites were analyzed: [U 13 C]-galactose, [U 13 C]-galactose-1-phosphate (Gal-1-P) and [ 13 C 6 ]- uridine diphosphate(UDP)-galactose. The ratio of [U 13 C]-Gal-1-P/ [ 13 C 6 ]-UDP-galactose was defined as the galactose index (GI)., Results: All patient cell lines could be distinguished from the control cell lines and there was a clear difference between variant and classical patients. Variant patients had lower levels of [U 13 C]-galactose and [U 13 C]-Gal-1-P than classical patients (though substantially higher than healthy controls) and higher levels of [ 13 C 6 ]-UDP-galactose than classical patients (though substantially lower than healthy controls) resulting in a different GI in all groups., Conclusions: GMP in fibroblasts is a sensitive method to determine residual galactose metabolism capacity, which can discriminate between patients with a classical presentation of galactosemia, patients with a variant presentation and healthy controls. GMP may be a useful method for early prognostication after further validation in a larger cohort of patients representing the full phenotypic spectrum of galactosemia.

In most organisms, galactose is metabolized via the Leloir pathway, which is conserved from bacteria to mammals. Utilization of galactose requires a close interplay of the metabolic enzymes, as misregulation or malfunction of individual components can lead to the accumulation of toxic intermediate compounds. For the phytopathogenic basidiomycete Ustilago maydis, galactose is toxic for wildtype strains, i.e. leads to growth repression despite the presence of favorable carbon sources as sucrose. The galactose sensitivity can be relieved by two independent modifications: (1) by disruption of Hxt1, which we identify as the major transporter for galactose, and (2) by a point mutation in the gene encoding the galactokinase Gal1, the first enzyme of the Leloir pathway. The mutation in gal1(Y67F) leads to reduced enzymatic activity of Gal1 and thus may limit the formation of putatively toxic galactose-1-phosphate. However, systematic deletions and double deletions of different genes involved in galactose metabolism point to a minor role of galactose-1-phosphate in galactose toxicity. Our results show that molecular triggers for galactose toxicity in U. maydis differ from yeast and mammals., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Metabolic dysregulation leading to sugar-phosphate accumulation is toxic in organisms ranging from bacteria to humans. By comparing two models of sugar-phosphate toxicity in Saccharomyces cerevisiae, we demonstrate that toxicity occurs, at least in part, through multiple, isomer-specific mechanisms, rather than a single general mechanism.

Human NUDT22 belongs to the diverse NUDIX family of proteins, but has, until now, remained uncharacterized. Here we show that human NUDT22 is a Mg 2+ -dependent UDP-glucose and UDP-galactose hydrolase, producing UMP and glucose 1-phosphate or galactose 1-phosphate. We present the structure of human NUDT22 alone and in a complex with the substrate UDP-glucose. These structures reveal a partially conserved NUDIX fold domain preceded by a unique N-terminal domain responsible for UDP moiety binding and recognition. The NUDIX domain of NUDT22 contains a modified NUDIX box identified using structural analysis and confirmed through functional analysis of mutants. Human NUDT22's distinct structure and function as a UDP-carbohydrate hydrolase establish a unique NUDIX protein subfamily., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

6-phospho-β-glucosidases and 6-phospho-β-galactosidases are enzymes that hydrolyze the β-glycosidic bond between a terminal non-reducing glucose-6-phosphate (Glc6P) or galactose-6-phosphate (Gal6P), respectively, and other organic molecules. Gan1D, a glycoside hydrolase (GH) belonging to the GH1 family, has recently been identified in a newly characterized galactan-utilization gene cluster in the bacterium Geobacillus stearothermophilus T-1. Gan1D has been shown to exhibit bifunctional activity, possessing both 6-phospho-β-galactosidase and 6-phospho-β-glucosidase activities. We report herein the complete 3D crystal structure of Gan1D, together with its acid/base catalytic mutant Gan1D-E170Q. The tertiary structure of Gan1D conforms well to the (β/α) 8 TIM-barrel fold commonly observed in GH enzymes, and its quaternary structure adopts a dimeric assembly, confirmed by gel-filtration and small-angle X-ray scattering results. We present also the structures of Gan1D in complex with the putative substrate cellobiose-6-phosphate (Cell6P) and the degradation products Glc6P and Gal6P. These complexes reveal the specific enzyme-substrate and enzyme-product binding interactions of Gan1D, and the residues involved in its glycone, aglycone, and phosphate binding sites. We show that the different ligands trapped in the active sites adopt different binding modes to the protein, providing a structural basis for the dual galactosidase/glucosidase activity observed for this enzyme. Based on this information, specific mutations were performed on one of the active site residues (W433), shifting the enzyme specificity from dual activity to a significant preference toward 6-phospho-β-glucosidase activity. These data and their comparison with structural data of related glucosidases and galactosidases are used for a more general discussion on the structure-function relationships in this sub-group of GH1 enzymes., Databases: Atomic coordinates of Gan1D-wild-type (WT)-P1, Gan1D-WT-C2, Gan1D-E170Q, Gan1D-WT-Gal6P, Gan1D-WT-Glc6P, and Gan1D-E170Q-Cell6P have been deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank, under accession codes 5OKB, 5OKJ/5OKH, 5OKA/5OK7, 5OKQ/5OKK, 5OKS/5OKR, and 5OKG/5OKE, respectively., (© 2017 Federation of European Biochemical Societies.)

Erwinia amylovora, a Gram-negative plant pathogen, is the causal agent of Fire Blight, a contagious necrotic disease affecting plants belonging to the Rosaceae family, including apple and pear. E. amylovora is highly virulent and capable of rapid dissemination in orchards; effective control methods are still lacking. One of its most important pathogenicity factors is the exopolysaccharide amylovoran. Amylovoran is a branched polymer made by the repetition of units mainly composed of galactose, with some residues of glucose, glucuronic acid and pyruvate. E. amylovora glucose-1-phosphate uridylyltransferase (UDP-glucose pyrophosphorylase, EC 2.7.7.9) has a key role in amylovoran biosynthesis. This enzyme catalyses the production of UDP-glucose from glucose-1-phosphate and UTP, which the epimerase GalE converts into UDP-galactose, the main building block of amylovoran. We determined EaGalU kinetic parameters and substrate specificity with a range of sugar 1-phosphates. At time point 120min the enzyme catalysed conversion of the sugar 1-phosphate into the corresponding UDP-sugar reached 74% for N-acetyl-α-d-glucosamine 1-phosphate, 28% for α-d-galactose 1-phosphate, 0% for α-d-galactosamine 1-phosphate, 100% for α-d-xylose 1-phosphate, 100% for α-d-glucosamine 1-phosphate, 70% for α-d-mannose 1-phosphate, and 0% for α-d-galacturonic acid 1-phosphate. To explain our results we obtained the crystal structure of EaGalU and augmented our study by docking the different sugar 1-phosphates into EaGalU active site, providing both reliable models for substrate binding and enzyme specificity, and a rationale that explains the different activity of EaGalU on the sugar 1-phosphates used. These data demonstrate EaGalU potential as a biocatalyst for biotechnological purposes, as an alternative to the enzyme from Escherichia coli, besides playing an important role in E. amylovora pathogenicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n = 240), with and without cumulus cells (CCs), were exposed for 4 hours to D-galactose (2 μM), galactitol (11 μM) and galactose 1-phosphate (0.1 mM), (corresponding to plasma concentrations in patients on galactose-restricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients.

Classic galactosemia (CG) is a potentially lethal inborn error of metabolism that results from the profound loss of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Neonatal detection and dietary restriction of galactose minimizes or resolves the acute sequelae of CG, but fails to prevent the long-term complications experienced by a majority of patients. One of the substrates of GALT, galactose-1-phosphate (Gal-1P), accumulates to high levels in affected infants, especially following milk exposure, and has been proposed as the key mediator of acute and long-term pathophysiology in CG. However, studies of treated patients demonstrate no association between red blood cell Gal-1P level and long-term outcome severity. Here, we used genetic, epigenetic and environmental manipulations of a Drosophila melanogaster model of CG to test the role of Gal-1P as a candidate mediator of outcome in GALT deficiency. Specifically, we both deleted and knocked down the gene encoding galactokinase (GALK) in control and GALT-null Drosophila, and assessed the acute and long-term outcomes of the resulting animals in the presence and absence of dietary galactose. GALK is the first enzyme in the Leloir pathway of galactose metabolism and is responsible for generating Gal-1P in humans and Drosophila Our data confirmed that, as expected, loss of GALK lowered or eliminated Gal-1P accumulation in GALT-null animals. However, we saw no concomitant rescue of larval survival or adult climbing or fecundity phenotypes. Instead, we saw that loss of GALK itself was not benign and in some cases phenocopied or exacerbated the outcome seen in GALT-null animals. These findings strongly contradict the long-standing hypothesis that Gal-1P alone underlies pathophysiology of acute and long-term outcomes in GALT-null Drosophila and suggests that other metabolite(s) of galactose, and/or other pathogenic factors, might be involved., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Ebosin produced by Streptomyces sp. 139 is a novel exopolysaccharide with anti-rheumatic arthritis activity in vivo and its biosynthesis gene cluster (ste) has been previously identified. In our previous research, ste5 gene has been identified as priming glycosyltransferase involved in Ebosin biosynthesis. However, it remains unclear how ste5 initiated Ebosin biosynthesis in molecular level. Here we show that Ebosin derivative produced by ste5 mutant lost the antagonist activities for IL-1R and Overexpression of ste5 in mutant dramatically enhanced the antagonist activities for IL-1R. For biochemical characterization of Ste5, the ste5 gene was cloned and expressed in Escherichia coli BL21. We identified that the recombinant Ste5 can transfer galactose-1-Phosphate (Gal-1-P) or glucose-1-Phosphate (Glc-1-P) from UDP-galactose and UDP-glucose to the lipid carrier located in the cytoplasmic membrane of Streptomyces sp. 139 (ste5(-)) with a continuous coupled spectrophotometric assay. 12.6 μM of Km was for UDP-galactose and 23.9 μM for UDP-glucose respectively. Our results indicate that Ste5 is bifunctional Gal-1-P and Glc-1-P transferase to initiate Ebosin biosynthesis and may be further applied in remoulding carbohydrate compounds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Classic galactosemia (CG) is a potentially lethal genetic disorder that results from profound loss of galactose-1-phosphate uridylyltransferase (GALT). CG is detected by newborn screening (NBS) in many countries; however, conclusive diagnosis can be complex due to broad and overlapping ranges of GALT activity. Molecular studies can also be complex due to allelic heterogeneity at the GALT locus., Methods: We conducted both biochemical and molecular follow-up studies for an infant flagged by NBS for possible galactosemia. To clarify the diagnosis we also conducted biochemical and RNA studies of lymphoblasts prepared from the child and one parent., Results: We identified a novel noncoding GALT variant, c.377+17C>T, that was homozygous in the child and heterozygous in both parents. The child and both parents also showed diminished GALT activity in red blood cells, and transformed lymphoblasts from the child and one parent further showed diminished GALT activity. However, qRT-PCR studies demonstrated apparently normal GALT mRNA levels in lymphoblasts, and Gal-1P values measured in the child following galactose exposure in infancy and at 1 year were normal., Conclusions: These results highlight the existence of rare but apparently benign variants in GALT and underscore the need for functional studies to distinguish pathogenic from benign variants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The ST0452 protein from the thermophilic archaean Sulfolobus tokodaii has been identified as an enzyme with multiple sugar-1-phosphate nucleotidylyltransferase and amino-sugar-1-phosphate acetyltransferase (amino-sugar-1-P AcTase) activities. Analysis of the protein showed that in addition to glucosamine-1-phosphate (GlcN-1-P) AcTase activity, it possesses unique galactosamine-1-phosphate (GalN-1-P) AcTase activity not detected in any other proteins. Comparison of the crystal structures of the ST0452 protein and GlmU from Escherichia coli (EcGlmU), which possesses only GlcN-1-P AcTase activity, showed that the overall sequence identity between these two proteins is less than 25 %, but the amino acid residues predicted to comprise the catalytic center of EcGlmU are conserved in the ST0452 protein. To understand the molecular mechanism by which the ST0452 amino-sugar-1-P AcTase activity recognizes two independent substrates, several ST0452 substitution and truncation mutant proteins were constructed and analyzed. We found that His308 is essential for both GalN-1-P and GlcN-1-P AcTase activities, whereas Tyr311 and Asn331 are important only for the GalN-1-P AcTase activity. In addition, deletion of the C-terminal 5 or 11 residues showed that the 11-residue C-terminal region exerts a modest stimulatory effect on GalN-1-P AcTase activity but dramatically suppresses GlcN-1-P AcTase activity. This region also appears to make an important contribution to the thermostability of the entire ST0452 protein. Systematic deletions from the C-terminus also demonstrated that the C-terminal region with the β-helix structure has an important role mediating the trimerization of the ST0452 protein. This is the first report of an analysis of a thermostable archaeal enzyme exhibiting multiple amino-sugar-1-P AcTase activities.

Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor., (Published by Elsevier Ltd.)

Background: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes., Results: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification., Conclusions: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides., General Significance: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The GNB/LNB (galacto-N-biose/lacto-N-biose) pathway plays a crucial role in bifidobacteria during growth on human milk or mucin from epithelial cells. It is thought to be the major route for galactose utilization in Bifidobacterium longum as it is an energy-saving variant of the Leloir pathway. Both pathways are present in B. bifidum, and galactose 1-phosphate (gal1P) is considered to play a key role. Due to its toxic nature, gal1P is further converted into its activated UDP-sugar through the action of poorly characterized uridylyltransferases. In this study, three uridylyltransferases (galT1, galT2, and ugpA) from Bifidobacterium bifidum were cloned in an Escherichia coli mutant and screened for activity on the key intermediate gal1P. GalT1 and GalT2 showed UDP-glucose-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.12), whereas UgpA showed promiscuous UTP-hexose-1-phosphate uridylyltransferase activity (EC 2.7.7.10). The activity of UgpA toward glucose 1-phosphate was about 33-fold higher than that toward gal1P. GalT1, as part of the bifidobacterial Leloir pathway, was about 357-fold more active than GalT2, the functional analog in the GNB/LNB pathway. These results suggest that GalT1 plays a more significant role than previously thought and predominates when B. bifidum grows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

The early lactation period of dairy cows, which produce high quantities of milk, is normally characterized by an insufficient energy intake to cover milk production and maintenance requirements. Mobilization of body reserves occurs to compensate this negative energy balance (NEB), and probably as a consequence there is a higher susceptibility to diseases and metabolic disorders. There are several diagnostic methods to detect NEB, usually involving ketosis related parameters. Due to the easy availability of milk this is a preferred matrix, but simple and robust predictors of NEB level are missing. To better understand the physiological mechanism of NEB, milk of cows subjected to different dry period lengths, in different energy balance status and lactation stage, were analyzed by untargeted metabolomics and proteomics techniques. Milk of cows in severe NEB showed higher concentrations of acute phase response proteins, unsaturated fatty acids, and galactose-1-phosphate. Improved energy balance (EB) resulted in higher concentration of cholesterol, cholesterol synthesis related proteins, and stomatin. The presence of stomatin and galactose-1-phosphate in milk was strongly dependent on the EB of the cows. These novel and interesting findings warrant more in-depth research to assess their applicability as robust indicators of NEB in milk and to clarify the role of stomatin and galactose-1-phophate in milk of dairy cows in NEB.

Galactosemia is an autosomal recessive disorder with a wide range of clinical abnormalities. Cellular oxidative stress is considered as one of the pathogenic mechanisms of galactosemia. In this study, we examined the activity of NADPH oxidase (NOX), a major superoxide-generating enzyme system, in peripheral blood lymphocytes (PBL) from galactosemia patients. PBL were isolated from galactosemia patients and healthy control subjects and used for cell culture studies and biochemical assays. PBL were cultured in the presence or absence of galactose or galactose-1-phosphate (Gal-1-P), and enzyme activities and/or gene expression of NOX, catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured in the cell homogenates. PBL isolated from galactosemia patients showed significantly reduced (P < 0.01) activities of catalase and GPx; however SOD activity remained unaltered. Galactosemia patients were found to have significantly (P < 0.01) increased levels of malondialdehyde (MDA) in blood lymphocytes. Enzymatic activity of NOX was significantly (P < 0.001) reduced in galactosemia patients; however, Western blotting revealed that NOX-1 protein was not significantly altered. Interestingly, levels of NOX activity in lymphocytes isolated from galactosemia patients significantly increased but remained subnormal when cultured in galactose-deficient medium for two weeks, indicating a galactose-mediated inhibition of NOX. Lymphocytes isolated from control subjects were found to have significantly (P < 0.01) reduced NOX activity when cultured in the presence of galactose or Gal-1-P for two weeks. These results show that galactose-induced cellular oxidative stress is not NOX mediated. However, impairment of the NOX system might be responsible for some of the clinical complications in galactosemia patients.

Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia.

Despite neonatal diagnosis and life-long dietary restriction of galactose, many patients with classic galactosemia grow to experience significant long-term complications. Among the more common are speech, cognitive, behavioral, ovarian and neurological/movement difficulties. Despite decades of research, the pathophysiology of these long-term complications remains obscure, hindering prognosis and attempts at improved intervention. As a first step to overcome this roadblock we have begun to explore long-term outcomes in our previously reported GALT-null Drosophila melanogaster model of classic galactosemia. Here we describe the first of these studies. Using a countercurrent device, a simple climbing assay, and a startle response test to characterize and quantify an apparent movement abnormality, we explored the impact of cryptic GALT expression on phenotype, tested the role of sublethal galactose exposure and galactose-1-phosphate (gal-1P) accumulation, tested the impact of age, and searched for potential anatomical defects in brain and muscle. We found that about 2.5% residual GALT activity was sufficient to reduce outcome severity. Surprisingly, sublethal galactose exposure and gal-1P accumulation during development showed no effect on the adult phenotype. Finally, despite the apparent neurological or neuromuscular nature of the complication we found no clear morphological differences between mutants and controls in brain or muscle, suggesting that the defect is subtle and/or is physiologic rather than structural. Combined, our results confirm that, like human patients, GALT-null Drosophila experience significant long-term complications that occur independently of galactose exposure, and serve as a proof of principle demonstrating utility of the GALT-null Drosophila model as a tool for exploring genetic and environmental modifiers of long-term outcome in GALT deficiency.

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Galactokinase (GalK), particularly GalK from Escherichia coli, has been widely employed for the synthesis of sugar-1-phosphates. In this study, a GalK from Bifidobacterium infantis ATCC 15697 (BiGalK) was cloned and over-expressed with a yield of over 80 mg/L cell cultures. The k(cat)/K(m) value of recombinant BiGalK toward galactose (164 s(-1) mM(-1)) is 296 times higher than that of GalK from E. coli, indicating that BiGalK is much more efficient in the phosphorylation of galactose. The enzyme also exhibits activity toward galacturonic acid, which has never been observed on other wild type GalKs. Further activity assays showed that BiGalK has broad substrate specificity toward both sugars and phosphate donors. These features make BiGalK an attractive candidate for the large scale preparation of galactose-1-phosphate and derivatives., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

L-Galactose-1-phosphate phosphatase (GPPase) is an enzyme involved in ascorbate biosynthesis in higher plants. We isolated a cDNA encoding GPPase from tobacco, and named it NtGPPase. The putative amino acid sequence of NtGPPase contained inositol monophosphatase motifs and metal binding sites. Recombinant NtGPPase hydrolyzed not only L-galactose-1-phosphate, but also myo-inositol-1-phosphate. The optimum pH for the GPPase activity of NtGPPase was 7.5. Its enzyme activity required Mg2+, and was inhibited by Li+ and Ca2+. Its fluorescence, fused with green fluorescence protein in onion cells and protoplasts of tobacco BY-2 cells, was observed in both the cytosol and nucleus. The expression of NtGPPase mRNA and protein was clearly correlated with L-ascorbic acid (AsA) contents of BY-2 cells during culture. The AsA contents of NtGPPase over expression lines were higher than those of empty lines at 13 d after subculture. This suggests that NtGPPase contributes slightly to AsA biosynthesis.

Reduced galactose 1-phosphate uridylyltransferase (GALT) activity is associated with the genetic disease type I galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GALT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (II) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GALT is required to assist greater understanding of the effects of disease-associated mutations., (Copyright © 2011 Wiley Periodicals, Inc.)

Human galactokinase (GALK) is the first enzyme in the Leloir pathway, converting α-d-galactose into galactose-1-phosphate (Gal-1-P). Recently, there is increasing interest in targeting GALK as a novel therapy to ameliorate the disease manifestations in patients with Classic Galactosemia as it would, in combination with (ga-)lactose restriction reduce accumulation of Gal-1-P, a cytotoxic agent. Previously, we identified 34 small molecule compounds that inhibited GALK in vitro using experimental high-throughput screening. In order to isolate useful lead compounds, we characterized these hits with regards to their kinase selectivity profiles, potency and capability to reduce Gal-1-P accumulation in patient cell lines, and their modes of action. We found that the majority of these compounds had IC(50)s ranging from 0.7μM to 33.3μM. When tested against other members of the GHMP kinase family, three compounds (1, 4, and 24) selectively inhibited GALK with high potency. Through alignment of GALK and mevalonate kinase (MVK) crystal structures, we identified that eight amino acid residues and an L1 loop were different within the ATP-binding pockets of these two closely related kinases. By site-directed mutagenesis experiments, we identified one amino acid residue required for the inhibitory function of two of the three selective compounds. Based on these results, we generated binding models of these two compounds using a high-precision docking program. Compounds 4 and 24 inhibited GALK in a mixed model, while compound 1 exhibited parabolic competitive inhibition. Most importantly, using cells from galactosemic patients we found that selected compounds lowered Gal-1-P concentrations., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Classic galactosemia is a potentially lethal disorder that results from profound impairment of galactose-1-phosphate uridylyltransferase (GALT). Despite decades of research, the underlying pathophysiology of classic galactosemia remains unclear, in part owing to the lack of an appropriate animal model. Here, we report the establishment of a Drosophila melanogaster model of classic galactosemia; this is the first whole-animal genetic model to mimic aspects of the patient phenotype. Analogous to humans, GALT-deficient D. melanogaster survive under conditions of galactose restriction, but accumulate elevated levels of galactose-1-phosphate and succumb during larval development following galactose exposure. As in patients, the potentially lethal damage is reversible if dietary galactose restriction is initiated early in life. GALT-deficient Drosophila also exhibit locomotor complications despite dietary galactose restriction, and both the acute and long-term complications can be rescued by transgenic expression of human GALT. Using this new Drosophila model, we have begun to dissect the timing, extent and mechanism(s) of galactose sensitivity in the absence of GALT activity.

UDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism.

Central dopaminergic (DA) systems are affected during human immunodeficiency virus (HIV) infection. So far, it is believed that they degenerate with progression of HIV disease because deterioration of DA systems is evident in advanced stages of infection. In this manuscript we found that (a) DA levels are increased and DA turnover is decreased in CSF of therapy-naïve HIV patients in asymptomatic infection, (b) DA increase does not modulate the availability of DA transporters and D2-receptors, (c) DA correlates inversely with CD4+ numbers in blood. These findings show activation of central DA systems without development of adaptive responses at DA synapses in asymptomatic HIV infection. It is probable that DA deterioration in advanced stages of HIV infection may derive from increased DA availability in early infection, resulting in DA neurotoxicity. Our findings provide a clue to the synergism between DA medication or drugs of abuse and HIV infection to exacerbate and accelerate HIV neuropsychiatric disease, a central issue in the neurobiology of HIV.

Background: Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)., Methods: [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed., Results: Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells., Conclusions: This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

Abundant in milk and other dairy products, lactose is considered to have an important role in oral microbial ecology and can contribute to caries development in both adults and young children. To better understand the metabolism of lactose and galactose by Streptococcus mutans, the major etiological agent of human tooth decay, a genetic analysis of the tagatose-6-phosphate (lac) and Leloir (gal) pathways was performed in strain UA159. Deletion of each gene in the lac operon caused various alterations in expression of a P(lacA)-cat promoter fusion and defects in growth on either lactose (lacA, lacB, lacF, lacE, and lacG), galactose (lacA, lacB, lacD, and lacG) or both sugars (lacA, lacB, and lacG). Failure to grow in the presence of galactose or lactose by certain lac mutants appeared to arise from the accumulation of intermediates of galactose metabolism, particularly galatose-6-phosphate. The glucose- and lactose-PTS permeases, EII(Man) and EII(Lac), respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIAB(Man), led to increased resistance to glucose-mediated CCR when lactose was used to induce the lac operon, but resulted in reduced lac gene expression in cells growing on galactose. Collectively, the results reveal a remarkably high degree of complexity in the regulation of lactose/galactose catabolism.

Background: The diagnosis of galactosemia usually involves the measurement of galactose-1-phosphate uridyltransferase (GALT) activity. Traditional radioactive and fluorescent GALT assays are nonspecific, laborious, and/or lack sufficient analytical sensitivity. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay for GALT enzyme activity measurement., Method: Our assay used stable isotope-labeled alpha- galactose-1-phosphate ([(13)C(6)]-Gal-1-P) as an enzyme substrate. Sample cleanup and separation were achieved by reversed-phase ion-pair chromatography, and the enzymatic product, isotope-labeled uridine diphosphate galactose ([(13)C(6)]-UDPGal), was detected by MS/MS at mass transition (571 > 323) and quantified by use of [(13)C(6)]-Glu-1-P (265 > 79) as an internal standard., Results: The method yielded a mean (SD) GALT enzyme activity of 23.8 (3.8) mumol x (g Hgb)(-1) x h(-1) in erythrocyte extracts from 71 controls. The limit of quantification was 0.04 micromol x (g Hgb)(-1) x h(-1) (0.2% of normal control value). Intraassay imprecision was determined at 4 different levels (100%, 25%, 5%, and 0.2% of the normal control values), and the CVs were calculated to be 2.1%, 2.5%, 4.6%, and 9.7%, respectively (n = 3). Interassay imprecision CVs were 4.5%, 6.7%, 8.2%, and 13.2% (n = 5), respectively. The assay recoveries at the 4 levels were higher than 90%. The apparent K(m) of the 2 substrates, Gal-1-P and UDPGlc, were determined to be 0.38 mmol/L and 0.071 mmol/L, respectively. The assay in erythrocytes of 33 patients with classical galactosemia revealed no detectable activity., Conclusions: This LC-MS/MS-based assay for GALT enzyme activity will be useful for the diagnosis and study of biochemically heterogeneous patients with galactosemia, especially those with uncommon genotypes and detectable but low residual activities.

Schizosaccharomyces species are currently the only known organisms with two types of genes encoding UDP-glucose/-galactose 4-epimerase, uge1(+) and gal10(+). A strain deleted for uge1(+) exhibited a severe galactosylation defect and a decrease in activity and in UDP-galactose content when grown in glucose-rich medium (2 % glucose), indicating that Uge1p is a major UDP-glucose/-galactose 4-epimerase under these growth conditions. In contrast, gal10(+) was efficiently expressed and involved in galactosylation of cell-surface proteins in low-glucose medium (0.1 % glucose and 2 % glycerol), but not in galactose-containing medium. In a uge1Deltagal10Delta strain, the galactosylation defect was suppressed and UDP-galactose content restored to wild-type levels in galactose-containing medium. Disruption of gal7(+), encoding galactose-1-phosphate uridylyltransferase, in the uge1Deltagal10Delta strain reversed suppression of the galactosylation defect and reduced levels of UDP-galactose, indicating that galactose is transported from the medium to the cytosol and is converted into UDP-galactose via galactose 1-phosphate by Gal7p in Sch. pombe.

The Leishmania parasite glycocalyx is rich in galactose-containing glycoconjugates that are synthesized by specific glycosyltransferases that use UDP-galactose as a glycosyl donor. UDP-galactose biosynthesis is thought to be predominantly a de novo process involving epimerization of the abundant nucleotide sugar UDP-glucose by the UDP-glucose 4-epimerase, although galactose salvage from the environment has been demonstrated for Leishmania major. Here, we present the characterization of an L. major UDP-sugar pyrophosphorylase able to reversibly activate galactose 1-phosphate into UDP-galactose thus proving the existence of the Isselbacher salvage pathway in this parasite. The ordered bisubstrate mechanism and high affinity of the enzyme for UTP seem to favor the synthesis of nucleotide sugar rather than their pyrophosphorolysis. Although L. major UDP-sugar pyrophosphorylase preferentially activates galactose 1-phosphate and glucose 1-phosphate, the enzyme is able to act on a variety of hexose 1-phosphates as well as pentose 1-phosphates but not hexosamine 1-phosphates and hence presents a broad in vitro specificity. The newly identified enzyme exhibits a low but significant homology with UDP-glucose pyrophosphorylases and conserved in particular is the pyrophosphorylase consensus sequence and residues involved in nucleotide and phosphate binding. Saturation transfer difference NMR spectroscopy experiments confirm the importance of these moieties for substrate binding. The described leishmanial enzyme is closely related to plant UDP-sugar pyrophosphorylases and presents a similar substrate specificity suggesting their common origin.

alpha-D-Galactose 1-phosphate (alphaGal1P) is an important building block for the synthesis of nucleotide sugars that are substrates for glycosyltransferases. We have previously reported the creation of novel lactose phosphorylase enzymes that are useful for the synthesis of alphaGal1P from the cheap and abundant lactose. Here we describe the application of such a lactose phosphorylase in a production system using permeabilized Escherichia coli cells. After purification of the product from the reaction mixture by anion-exchange chromatography and ethanol precipitation, 9.5 grams of highly pure alphaGal1P were obtained from a 1 l reaction volume.

Pregnancy, delivery, and postpartal metabolic control was monitored biochemically in five patients (22-38 years of age) with clinically, enzymatically, and genotypically established classical galactosaemia and good dietary compliance. Three of the patients performed breast feeding of their newborns. Monitoring parameters were galactose-1-phosphate and galactitol concentrations in erythrocytes and urinary excretion of galactose, galactitol, galactonate, and lactose. During pregnancy, a small but steady increase of renal metabolite excretion rates was observed. After delivery, a moderate transient increase of metabolite concentrations with peak values within the first week post partum occurred, irrespective of breast feeding. Altogether, there was no evidence for clinically or subclinically significant changes of metabolic control during pregnancy, delivery, or lactation. In conclusion, a specific metabolic monitoring is apparently not required in pregnant galactosemic women, and breast feeding of the nongalactosemic offspring can be recommended.

The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of alpha-D-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer.

In Saccharomyces cerevisiae, lithium induces a 'galactosemia-like' phenotype as a consequence of inhibition of phosphoglucomutase, a key enzyme in galactose metabolism. Induced galactose toxicity is prevented by deletion of GAL4, which inhibits the transcriptional activation of genes involved in galactose metabolism and by deletion of the galactokinase (GAL1), indicating that galactose-1-phosphate, a phosphorylated intermediate of the Leloir pathway, is the toxic compound. As an alternative to inhibiting entry and metabolism of galactose, we investigated whether deviation of galactose metabolism from the Leloir pathway would also overcome the galactosemic effect of lithium. We show that cells overexpressing the aldose reductase GRE3, which converts galactose to galactitol, are more tolerant to lithium than wild-type cells when grown in galactose medium and they accumulate more galactitol and less galactose-1-phosphate. Overexpression of GRE3 also suppressed the galactose growth defect of the 'galactosemic'gal7- and gal10-deleted strains, which lack galactose-1-P-uridyltransferase or UDP-galactose-4-epimerase activities, respectively. Furthermore, the effect of GRE3 was independent of the inositol monophosphatases INM1 and INM2. We propose that lithium induces a galactosemic state in yeast and that inhibition of the Leloir pathway before the phosphorylation step or stimulation of galactitol production suppresses lithium-induced galactose toxicity.

A beta-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of alpha-d-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Galbeta1 --> 3GlcNAc) and galacto-N-biose (GNB) (Galbeta1 --> 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k(cat)/K(m) for LNB was approximately 60 times higher than the k(cat)/K(m) for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/lacto-N-biose I phosphorylases.

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.

The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.