Leloir Pathway Of Galactose Metabolism Research Articles

Restoring galactose metabolism without restoring GALT rescues both compromised survival in larvae and an adult climbing deficit in a GALT-null D. Melanogaster model of classic galactosemia.

Classic galactosemia (CG) is an autosomal recessive disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the middle enzyme in the highly conserved Leloir pathway of galactose metabolism. That galactose metabolism is disrupted in patients with CG, and in GALT-null microbial, cell culture, and animal models of CG, has been known for many years. However, whether the long-term developmental complications of CG result from disrupted galactose metabolism alone, or from loss of some independent moonlighting function of GALT, in addition to disrupted galactose metabolism, has been posed but never resolved. Here, we addressed this question using a GALT-null Drosophila melanogaster model of CG engineered to express uridine diphosphate (UDP)-glucose/galactose pyrophosphorylase (UGGP), a plant enzyme that effectively bypasses GALT in the Leloir pathway by converting substrates uridine triphosphate (UTP) plus galactose-1-phosphate (gal-1P) into products UDP-galactose plus pyrophosphate (PPi). While GALT and UGGP share one substrate (gal-1P) and one product (UDP-galactose), they are structurally and evolutionarily unrelated enzymes. It is therefore extremely unlikely that they would also share a moonlighting function. We found that GALT-null flies expressing UGGP showed not only partial rescue of metabolic abnormalities and acute larval sensitivity to dietary galactose, as expected, but also full rescue of an adult motor deficit otherwise seen in this model. By extension, these results may offer insights to the underlying bases of at least some acute and long-term complications experienced by patients with CG.

Effects of Bifidobacterium longum CCFM5871 as an adjunct starter culture on the production of fermented milk

Bifidobacterium has been widely studied for its probiotic properties. In our previous study, Bifidobacterium longum CCFM5871 (CCFM5871) showed potential to relieve inflammation and constipation. In current study, we investigated the application of CCFM5871 as an adjunct starter culture for the production of fermented milk. First, genome analysis indicated that CCFM5871 possess various carbohydrate metabolic pathways, including the Leloir pathway of galactose metabolism and galactose related genes GH2 and GH27. Comparative genomic analysis shows that similar functional gene were detected in other bifidobacteria strains applied in dairy fermentation. Then the sugar metabolism phenotype experiment verified the result of genomic analysis. Subsequently, we evaluated the effects of CCFM5871 supplementation on the production and storage of fermented milk by monitoring changes in multiple parameters over 14 days of storage at 4 °C, including pH, titratable acidity, physical properties, volatile flavor compounds, and sensory quality. The bacterial level was significantly higher at the fermentation termination than the fermentation initiation, indicating that CCFM5871 can be regarded as a candidate for adjunct start culture. Compared to the control group, supplementation with CCFM5871 increased acidification and solidification of fermented milk. Moreover, we revealed that there was no other significant difference between the yogurt fermented with CCFM5871 and the yogurt with culture only in flavor and taste. In summary, our results identified that CCFM5871 may be an appropriate co-culture for fermented milk, and the genomic analysis may be instructive for future to filter the culture in fermented milk.

Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues.

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring galacose-1-phosphate uridyltransferase activity in cell culture and animal tissues.

CONTRASTING TOXICITY OF D-GALACTOSE ON OOCYTES AND EMBRYOS

A rare mutation in galactose-1-phosphate uridyltransferase, an enzyme in the Leloir pathway of galactose metabolism, causes classic galactosemia (CG), a metabolic disorder characterized by harmful accumulation of D-galactose and its metabolites galactose 1-phosphate (gal 1-P) and galactitol in physiological systems. One especially susceptible system is the female reproductive tract, specifically the ovaries, which often leads to primary ovarian insufficiency (POI) even with careful monitoring of diet. However, some number of pregnancies in CG patients has been reported even though the general perception is that they are infertile. Previous studies have delineated a mechanism of D-galactose toxicity in oocytes associated with apoptosis and excessive reactive oxygen species, but considering the incidence of pregnancy in CG patients, embryos could be less affected by D-galactose toxicity. Therefore, the aim of this study is to explicate the effects of D-galactose and its metabolites on embryos and subsequent development. This was an experimental case-control design. D-galactose (2 mM), galactitol (11 mM) and gal 1-P (0.1 mM), with concentrations corresponding to those typically found in plasma of CG patients under dietary restrictions, were used to treat one-cell mouse embryos (n = 31) for 4 h. After exposure, the embryos were then incubated in culture for 120 h. Similar procedures were used to treat metaphase II mouse oocytes (n = 120), which were then fertilized in vitro, and then cultured for 120 h. Cells were analyzed for development at 24, 48, and 96 h. Cleavage stage embryos were assessed for nuclear fragments, morphology, and blastomere count. Blastocyte stage embryos were assessed for blastocoel expansion, trophectoderm morphology, and inner cell mass quality. No differences were found between directly treated embryos and untreated controls. Conversely, there was significant decline in cleavage and blastocyst development in embryos derived from treated, fertilized oocytes compared to untreated controls. Considering the relatively high pregnancy rate observed in women with CG-associated POI compared to all women with POI, D-galactose exposure does not appear to be embryotoxic. Thus, oocytes that avoid the harmful effects of D-galactose or its metabolites have the potential for a normal developmental course even if they are then later exposed to D-galactose or its metabolites. These results should encourage women with CG to maintain optimism for childbearing and childbirth.

Low-sugar yogurt making by the co-cultivation of Lactobacillus plantarum WCFS1 with yogurt starter cultures

Yogurt making traditionally relies on the simultaneous utilization of 2 starters: Streptococcus thermophilus and Lactobacillus bulgaricus, but these 2 strains normally metabolize the glucose portion of lactose and release galactose into extracellular medium, resulting in high levels of residual galactose and unfermented lactose in yogurt, which leads to several industrial and health concerns. In this study, we found that Lactobacillus plantarum could effectively metabolize both lactose and galactose. Comparative genomic analysis demonstrated the constant presence of a chromosome-encoded Leloir pathway for galactose metabolism in Lb. plantarum species, and the gal operon was driven by a strong constitutive promoter in Lb. plantarum WCFS1, displaying great potential in low-sugar yogurt making. To test this hypothesis, Lb. plantarum WCFS1 was co-cultured with S. thermophilus or Lb. bulgaricus in lactose-based medium. Results showed that lactose was consumed completely and galactose was metabolized efficiently. For yogurt making, co-cultivation of Lb. plantarum WCFS1 with yogurt starter cultures produced a higher reduction of total sugar content compared with the traditional fermentation processes. In addition, the sensory analysis indicated that the yogurt fermented with yogurt starter cultures and Lb. plantarum WCFS1 was acceptable to consumers in appearance, texture, and flavor. Therefore, this study emphasized the potential to manufacture low-sugar yogurt by the co-cultivation of Lb. plantarum with yogurt starter cultures.

A galactose-1-phosphate uridylyltransferase-null rat model of classic galactosemia mimics relevant patient outcomes and reveals tissue-specific and longitudinal differences in galactose metabolism.

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.

Hepatic GALE Regulates Whole-Body Glucose Homeostasis by Modulating Tff3 Expression.

Transcripts of key enzymes in the Leloir pathway of galactose metabolism in mouse livers are significantly increased after chronic high-fat/high-sucrose feeding. UDP-galactose-4-epimerase (GALE) is the last enzyme in this pathway that converts UDP-galactose to UDP-glucose and was previously identified as a downstream target of the endoplasmic reticulum (ER) stress effector spliced X-box binding protein 1, suggesting an interesting cross talk between galactose and glucose metabolism in the context of hepatic ER stress and whole-body metabolic fitness. However, its specific role in glucose metabolism is not established. Using an inducible and tissue-specific mouse model, we report that hepatic overexpression of Gale increases gluconeogenesis from pyruvate and impairs glucose tolerance. Conversely, genetic reduction of Gale in liver improves glucose tolerance. Transcriptional profiling identifies trefoil factor 3 (Tff3) as one of the downstream targets of GALE. Restoration of Tff3 expression corrects glucose intolerance in Gale-overexpressing mice. These studies reveal a new link between hepatic GALE activity and whole-body glucose homeostasis via regulation of hepatic Tff3 expression.

Drosophila melanogaster Models of Galactosemia.

The galactosemias are a family of autosomal recessive genetic disorders resulting from impaired function of the Leloir pathway of galactose metabolism. Type I, or classic galactosemia, results from profound deficiency of galactose-1-phosphate uridylyltransferase, the second enzyme in the Leloir pathway. Type II galactosemia results from profound deficiency of galactokinase, the first enzyme in the Leloir pathway. Type III galactosemia results from partial deficiency of UDP galactose 4'-epimerase, the third enzyme in the Leloir pathway. Although at least classic galactosemia has been recognized clinically for more than 100 years, and detectable by newborn screening for more than 50 years, all three galactosemias remain poorly understood. Early detection and dietary restriction of galactose prevent neonatal lethality, but many affected infants grow to experience a broad range of developmental and other disabilities. To date, there is no intervention known that prevents or reverses these long-term complications. Drosophila melanogaster provides a genetically and biochemically facile model for these conditions, enabling studies that address mechanism and open the door for novel approaches to intervention.

Acute and long-term outcomes in a Drosophila melanogaster model of classic galactosemia occur independently of galactose-1-phosphate accumulation.

ABSTRACTClassic 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.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts

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 RT2 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.

Characterization of hypothetical protein VNG0128C from Halobacterium NRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism

VNG0128C, a hypothetical protein from Halobacterium NRC-1, was chosen for detailed insilico and experimental investigations. Computational exercises revealed that VNG0128C functions as NAD+ binding protein. The phylogenetic analysis with the homolog sequences of VNG0128C suggested that it could act as UDP-galactose 4-epimerase. Hence, the VNG0128C sequence was modeled using a suitable template and docking studies were performed with NAD and UDP-galactose as ligands. The binding interactions strongly indicate that VNG0128C could plausibly act as UDP-galactose 4-epimerase. In order to validate these insilico results, VNG0128C was cloned in pUC57, subcloned in pET22b+, expressed in BL21 cells and purified using nickel affinity chromatography. An assay using blue dextran was performed to confirm the presence of NAD binding domain. To corroborate the epimerase like enzymatic role of the hypothetical protein, i.e. the ability of the enzyme to convert UDP-galactose to UDP-glucose, the conversion of NAD to NADH was measured. The experimental assay significantly correlated with the insilico predictions, indicating that VNG0128C has a NAD+ binding domain with epimerase activity. Consequently, its key role in nucleotide-sugar metabolism was thus established. Additionally, the work highlights the need for a methodical characterization of hypothetical proteins (less studied class of biopolymers) to exploit them for relevant applications in the field of biology.

A De Novo Variant in Galactose-1-P Uridylyltransferase (GALT) Leading to Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal genetic disease that results from profound impairment of galactose-1-P uridylyltransferase (GALT), the middle enzyme in the Leloir pathway of galactose metabolism. Patients with CG carry pathogenic loss-of-function mutations in both of their GALT alleles; the parents of patients are considered obligate carriers. We report here a first exception to that rule - a de novo GALT variant in a patient with classic galactosemia. The new variant, c.563A>C (p.Q188P), which introduces a missense substitution near the active site of the GALT enzyme, was found in the compound heterozygous state in a child with classic galactosemia, but not in either of her parents. Extensive genomic studies of DNA from the child and both parents confirmed the expected degrees of relationship in the trio as well as inheritance of a common c.563A>G (p.Q188R) GALT mutation from the mother. This result demonstrates that not all pathogenic GALT mutations are inherited and raises concern that GALT may have a higher new mutation rate than previously believed.

Cryptic residual GALT activity is a potential modifier of scholastic outcome in school age children with classic galactosemia

Classic galactosemia is a potentially lethal disorder that results from profound deficiency of galactose-1-phosphate uridylyltransferase (GALT), the second enzyme in the Leloir pathway of galactose metabolism. Although early diagnosis and rigorous dietary restriction of galactose prevent or resolve the potentially lethal acute symptoms, patients are at markedly increased risk of long-term complications including significant cognitive, speech, and behavioral difficulties, among other problems. The mechanisms that underlie these long-term complications remain unclear, as do the factors that modify their severity. Here we explored the scholastic and behavioral outcomes experienced by a cohort of 54 school age children with classic galactosemia. Data collected included survey responses from parents and teachers, school records including standardized test scores, and GALT genotype data used to estimate predicted residual GALT activity based on a yeast expression system. As expected, many but not all of the children in our study demonstrated speech, scholastic, and behavioral difficulties. Perhaps most striking, we found that predicted cryptic residual GALT activity, often below the threshold of detection of clinical assays, appeared to modify scholastic outcome. These data raise the intriguing possibility that cryptic GALT activity might also influence the severity of other long-term complications in classic galactosemia.

Engineered Escherichia coli for simultaneous utilization of galactose and glucose

In this study, the Leloir pathway of galactose metabolism was rebuilt in Escherichia coli to remove CCR and amplify galactose utilization rate. All genes encoding pathway enzymes were expressed under the control of a synthetic module that included promoters, 5′-untranslated regions, and terminators as a re-organized single operon in the chromosome. The engineered strain showed both an enhanced galactose utilization rate and the capacity to simultaneously assimilate galactose and glucose. This work demonstrates the feasibility of using synthetic biology tools to re-build biological systems for engineering purpose.

UDP-Galactose 4′-Epimerase Activities toward UDP-Gal and UDP-GalNAc Play Different Roles in the Development of Drosophila melanogaster

In both humans and Drosophila melanogaster, UDP-galactose 4′-epimerase (GALE) catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc) in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-acetylglucosamine (UDP-glcNAc). All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE) efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU) efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.

TRANSLATIONAL IMPACT

UDP-galactose 4’ epimerase (GALE) plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose when exogenous sources are lacking, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. GALE catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway of galactose metabolism and, in humans, also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. No patients completely lacking GALE activity have been found, suggesting that loss of GALE must be lethal. However, partial loss of GALE can be frequent, at least in some populations, and results in epimerase deficiency galactosemia. This spectrum disorder ranges in severity from benign to potentially lethal, depending, among other factors, on the degree and tissue-specificity of GALE impairment. The prognosis and treatment for patients with epimerase deficiency galactosemia are currently ill-defined, partly because affected individuals are often either missed or misdiagnosed, but also owing to the lack of an appropriate animal model.This paper reports the generation and initial characterization of the first whole-animal model of GALE deficiency, using the fruit fly Drosophila melanogaster. Drosophila lacking GALE died as embryos, confirming that GALE function is essential in developing animals. Larvae in which Drosophila GALE (dGALE) expression was conditionally inhibited by dGALE-specific RNA interference (RNAi) also died within days of GALE loss. Both types of affected embryos were rescued by expression of a human GALE (hGALE) transgene, showing the specificity of the mutation. To examine possible tissue specificity of the lethal GALE phenotype, conditional knockdown and hGALE transgene expression were used to show that GALE expression in the gut primordium and Malpighian tubules was both necessary and sufficient for Drosophila survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with a partial loss of GALE activity survived in the absence of galactose, but succumbed during development if exposed to dietary galactose.These data establish the utility of the fly model of generalized GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

Computer-Aided Identification ofTrypanosoma bruceiUridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness

Trypanosoma brucei, the causative agent of human African trypanosomiasis, affects tens of thousands of sub-Saharan Africans. As current therapeutics are inadequate due to toxic side effects, drug resistance, and limited effectiveness, novel therapies are urgently needed. UDP-galactose 4′-epimerase (TbGalE), an enzyme of the Leloir pathway of galactose metabolism, is one promising T. brucei drug target. We here use the relaxed complex scheme, an advanced computer-docking methodology that accounts for full protein flexibility, to identify inhibitors of TbGalE. An initial hit rate of 62% was obtained at 100 μM, ultimately leading to the identification of 14 low-micromolar inhibitors. Thirteen of these inhibitors belong to a distinct series with a conserved binding motif that may prove useful in future drug design and optimization.

Galactose toxicity in animals

In most organisms, productive utilization of galactose requires the highly conserved Leloir pathway of galactose metabolism. Yet, if this metabolic pathway is perturbed due to congenital deficiencies of the three associated enzymes, or an overwhelming presence of galactose, this monosaccharide which is abundantly present in milk and many non-dairy foodstuffs, will become highly toxic to humans and animals. Despite more than four decades of intense research, little is known about the molecular mechanisms of galactose toxicity in human patients and animal models. In this contemporary review, we take a unique approach to present an overview of galactose toxicity resulting from the three known congenital disorders of galactose metabolism and from experimental hypergalactosemia. Additionally, we update the reader about research progress on animal models, as well as advances in clinical management and therapies of these disorders.

Molecular cloning, sequence analysis and homology modelling of galE encoding UDP galactose 4­ epimerase of Aeromonas hydrophila

A. hydrophila, a ubiquitous gram-negative bacterium present in aquatic environments, has been implicated in illness in humans, fish and amphibians. Lipopolysaccharides (LPS), a surface component of the outer membrane, are one of the main virulent factors of gram-negative bacteria. UDP-galactose 4-epimerase (GalE) catalyses the last step in the Leloir pathway of galactose metabolism and provides precursor for the biosynthesis of extracellular LPS and capsule. Due to its key role in LPS biosynthesis, it is a potential drug target. The present study describes cloning, sequence analysis and prediction of three dimensional structure of the deduced amino acid sequence of the galE of A. hydrophila AH17. The cloned galE consists of the putative promoter-operator region, and an open reading frame of 338 amino acid residues. Sequence alignment and predicted 3Dstructure revealed that the GalE of A. hydrophila consists of the signature sequences of the epimerase super family. The present study reports the molecular modeling / 3D-structure prediction of GalE of A. hydrophila. Further, the potential regions of the enzyme that can be targeted for drug design are identified.

Pleiotropy and GAL pathway degeneration in yeast

Traits that do not contribute to fitness are expected to be lost during the course of evolution, either as a result of selection or drift. The Leloir pathway of galactose metabolism (GAL) is an extensively studied metabolic pathway that degenerated on at least three independent occasions during the evolutionary diversification of yeasts, suggesting that the pathway is costly to maintain in environments that lack galactose. Here I test this hypothesis by competing GAL pathway deletion mutants of Saccharomyces cerevisiae against an isogenic strain with an intact GAL pathway under conditions where expression of the pathway is normally induced, repressed, or uninduced. These experiments do not support the hypothesis that pleiotropy drives GAL pathway degeneration, because mutations that knock out individual GAL genes do not tend to increase fitness in the absence of galactose. At a molecular level, this result can be explained by the fact that yeast uses inexpensive regulatory proteins to tightly regulate the expression of structural genes that are costly to express. I argue that these results have general relevance for our understanding of the fitness consequences of gene disruption in yeast.