Journal articles: 'Leloir pathway'

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Sharpe, Martyn A., Omkar B. Ijare, David S. Baskin, Alexandra M. Baskin, Brianna N. Baskin, and Kumar Pichumani. "The Leloir Cycle in Glioblastoma: Galactose Scavenging and Metabolic Remodeling." Cancers 13, no. 8 (April 10, 2021): 1815. http://dx.doi.org/10.3390/cancers13081815.

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Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The 13C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

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van den Brink, J., M. Akeroyd, R. van der Hoeven, J. T. Pronk, J. H. de Winde, and P. Daran-Lapujade. "Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose–galactose transitions." Microbiology 155, no. 4 (April 1, 2009): 1340–50. http://dx.doi.org/10.1099/mic.0.025775-0.

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Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose–galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose–galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

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Brophy, Megan L., John E. Murphy, and Robert D. Bell. "Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues." Journal of Biological Methods 8, no. 2 (June 29, 2021): e149. http://dx.doi.org/10.14440/jbm.2021.355.

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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.

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Thoden, J. B., and H. M. Holden. "Structural studies of the enzymes of the Leloir pathway." Acta Crystallographica Section A Foundations of Crystallography 58, s1 (August 6, 2002): c94. http://dx.doi.org/10.1107/s0108767302088785.

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De Bruyn, Frederik, Joeri Beauprez, Jo Maertens, Wim Soetaert, and Marjan De Mey. "Unraveling the Leloir Pathway of Bifidobacterium bifidum: Significance of the Uridylyltransferases." Applied and Environmental Microbiology 79, no. 22 (September 6, 2013): 7028–35. http://dx.doi.org/10.1128/aem.02460-13.

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ABSTRACTThe 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 inBifidobacterium longumas it is an energy-saving variant of the Leloir pathway. Both pathways are present inB. 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, andugpA) fromBifidobacterium bifidumwere cloned in anEscherichia colimutant 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 whenB. bifidumgrows on lactose and human milk oligosaccharides. GalT2 activity is required only during growth on substrates with a GNB core such as mucin glycans.

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Bettenbrock, Katja, and Carl-Alfred Alpert. "The gal Genes for the Leloir Pathway ofLactobacillus casei 64H." Applied and Environmental Microbiology 64, no. 6 (June 1, 1998): 2013–19. http://dx.doi.org/10.1128/aem.64.6.2013-2019.1998.

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ABSTRACT The gal genes from the chromosome ofLactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of theL. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking theHindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKppromoter is inducible by galactose in the medium, whilegalEp constitutes a semiconstitutive promoter located ingalK.

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Sharpe, Martyn, Alexandra Baskin, Brianna Baskin, David Baskin, and Sudhir Raghavan. "DDRE-17. TARGETING GLIOBLASTOMA’S GALACTOSE SCAVENGING PATHWAY." Neuro-Oncology 23, Supplement_6 (November 2, 2021): vi78. http://dx.doi.org/10.1093/neuonc/noab196.301.

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Abstract BACKGROUND We have recently shown that GBM use D-galactose (Gal) as a substrate, in vitro and in vivo. Gal is imported via Glut3 and/or Glut14 and metabolized through the Leloir pathway. We investigated 4-deoxy-4-fluorogalactose (4DFG) as the lead compound in a family of galactose-based antimetabolites. 4DFG is a potent chemotherapeutic in monotherapy and can bolster existing therapies. METHODS We examined the alteration of glioma metabolism in vitro and in vivo induced by 4DFG. 1H/13C-NMR and optical probes were used to interrogate the effects of 4DFG on glycolysis and mitochondrial respiration in primary glioma cell cultures. Labeled lectins were used to assay for the disruption of glycan synthesis induced by 4DFG. An intracranial model of primary GBM was used to assess efficacy and toxicity in vivo. RESULTS NMR reveals that at physiological concentrations of glucose, low concentrations of 4DFG (5 μM) is able to inhibit glycolytic and mitochondrial flux by approximately 12%, p< 0.05. Analysis using lectins shows a collapse in general glycan synthesis, but most especially in the incorporation of both Gal and GalNAc sugars. In nude mice with intracranial primary GBM, six treatments of 4DFG increased survival from 23 to 50 days, p< 0.002. DISCUSSION The ability of GBM to scavenge galactose allows us to target the Glut3/14 import and Leloir metabolic pathway using galactose-based anti-metabolites. Our first-generation compound is highly effective as a monotherapy, inhibiting glucose metabolism and glycan synthesis.

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Abranches, Jacqueline, Yi-Ywan M. Chen, and Robert A. Burne. "Galactose Metabolism by Streptococcus mutans." Applied and Environmental Microbiology 70, no. 10 (October 2004): 6047–52. http://dx.doi.org/10.1128/aem.70.10.6047-6052.2004.

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ABSTRACT The galK gene, encoding galactokinase of the Leloir pathway, was insertionally inactivated in Streptococcus mutans UA159. The galK knockout strain displayed only marginal growth on galactose, but growth on glucose or lactose was not affected. In strain UA159, the sugar phosphotransferase system (PTS) for lactose and the PTS for galactose were induced by growth in lactose and galactose, although galactose PTS activity was very low, suggesting that S. mutans does not have a galactose-specific PTS and that the lactose PTS may transport galactose, albeit poorly. To determine if the galactose growth defect of the galK mutant could be overcome by enhancing lactose PTS activity, the gene encoding a putative repressor of the operon for lactose PTS and phospho-β-galactosidase, lacR, was insertionally inactivated. A galK and lacR mutant still could not grow on galactose, although the strain had constitutively elevated lactose PTS activity. The glucose PTS activity of lacR mutants grown in glucose was lower than in the wild-type strain, revealing an influence of LacR or the lactose PTS on the regulation of the glucose PTS. Mutation of the lacA gene of the tagatose pathway caused impaired growth in lactose and galactose, suggesting that galactose can only be efficiently utilized when both the Leloir and tagatose pathways are functional. A mutation of the permease in the multiple sugar metabolism operon did not affect growth on galactose. Thus, the galactose permease of S. mutans is not present in the gal, lac, or msm operons.

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Grossiord, Benoît, Elaine E. Vaughan, Evert Luesink, and Willem M. de Vos. "Genetics of galactose utilisation via the Leloir pathway in lactic acid bacteria." Le Lait 78, no. 1 (1998): 77–84. http://dx.doi.org/10.1051/lait:1998110.

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Holden, Hazel M., Ivan Rayment, and James B. Thoden. "Structure and Function of Enzymes of the Leloir Pathway for Galactose Metabolism." Journal of Biological Chemistry 278, no. 45 (August 15, 2003): 43885–88. http://dx.doi.org/10.1074/jbc.r300025200.

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Thoden, James B., and Hazel M. Holden. "The Molecular Architecture of Galactose Mutarotase/UDP-Galactose 4-Epimerase from Saccharomyces cerevisiae." Journal of Biological Chemistry 280, no. 23 (March 28, 2005): 21900–21907. http://dx.doi.org/10.1074/jbc.m502411200.

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The metabolic pathway by which β-d-galactose is converted to glucose 1-phosphate is known as the Leloir pathway and consists of four enzymes. In most organisms, these enzymes appear to exist as soluble entities in the cytoplasm. In yeast such as Saccharomyces cerevisiae, however, the first and last enzymes of the pathway, galactose mutarotase and UDP-galactose 4-epimerase, are contained within a single polypeptide chain referred to as Gal10p. Here we report the three-dimensional structure of Gal10p in complex with NAD+, UDP-glucose, and β-d-galactose determined to 1.85-Å resolution. The enzyme is dimeric with dimensions of ∼91 Å × 135 Å × 108 Å and assumes an almost V-shaped appearance. The overall architecture of the individual subunits can be described in terms of two separate N- and C-terminal domains connected by a Type II turn formed by Leu-357 to Val-360. The first 356 residues of Gal10p fold into the classical bilobal topology observed for all other UDP-galactose 4-epimerases studied thus far. This N-terminal domain contains the binding sites for NAD+ and UDP-glucose. The polypeptide chain extending from Glu-361 to Ser-699 adopts a β-sandwich motif and harbors the binding site for β-d-galactose. The two active sites of Gal10p are separated by over 50 Å. This investigation represents the first structural analysis of a dual function enzyme in the Leloir pathway.

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Barreto, Marlen, Eugenia Jedlicki, and David S. Holmes. "Identification of a Gene Cluster for the Formation of Extracellular Polysaccharide Precursors in the Chemolithoautotroph Acidithiobacillus ferrooxidans." Applied and Environmental Microbiology 71, no. 6 (June 2005): 2902–9. http://dx.doi.org/10.1128/aem.71.6.2902-2909.2005.

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ABSTRACT A cluster of five genes, proposed to be involved in the formation of extracellular polysaccharide (EPS) precursors via the Leloir pathway, have been identified in the acidophilic autotroph Acidithiobacillus ferrooxidans. The order of the genes is luxA-galE-galK-pgm-galM, encoding a LuxA-like protein, UDP-glucose 4-epimerase, galactokinase, phosphoglucomutase, and galactose mutarotase, respectively. The gal cluster forms a single transcriptional unit and is therefore an operon. Two other putative genes of the Leloir pathway, galU, potentially encoding UDP-glucose pyrophosphorylase, and a gene designated galT-like, which may encode a galactose-1-phosphate uridylyltransferase-like activity, were found unlinked in the genome. Using semiquantitative reverse transcription-PCR, the genes of the gal operon were shown to be expressed more during growth in iron medium than in growth in sulfur medium. The functions of galE, pgm, galU, and the galT-like gene were validated by complementation of Escherichia coli mutants and by in vitro enzyme assays. The data suggest that A. ferrooxidans is capable of synthesizing the EPS precursors UDP-glucose and UDP-galactose. In addition, genes rfbA, -B, -C, and -D were identified in the genome of A. ferrooxidans, suggesting that it can also synthesize the EPS precursor dTDP-rhamnose. Since EPSs constitute the major bulk of biofilms, this study may provide an initial model for the metabolic pathways involved in biofilm formation in A. ferrooxidans and aid in understanding the role of biofilms in mineral leaching and the formation of acid mine drainage.

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Grossiord, Benoît P., Evert J. Luesink, Elaine E. Vaughan, Alain Arnaud, and Willem M. de Vos. "Characterization, Expression, and Mutation of the Lactococcus lactis galPMKTE Genes, Involved in Galactose Utilization via the Leloir Pathway." Journal of Bacteriology 185, no. 3 (February 1, 2003): 870–78. http://dx.doi.org/10.1128/jb.185.3.870-878.2003.

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ABSTRACT A cluster containing five similarly oriented genes involved in the metabolism of galactose via the Leloir pathway in Lactococcus lactis subsp. cremoris MG1363 was cloned and characterized. The order of the genes is galPMKTE, and these genes encode a galactose permease (GalP), an aldose 1-epimerase (GalM), a galactokinase (GalK), a hexose-1-phosphate uridylyltransferase (GalT), and a UDP-glucose 4-epimerase (GalE), respectively. This genetic organization reflects the order of the metabolic conversions during galactose utilization via the Leloir pathway. The functionality of the galP, galK, galT, and galE genes was shown by complementation studies performed with both Escherichia coli and L. lactis mutants. The GalP permease is a new member of the galactoside-pentose-hexuronide family of transporters. The capacity of GalP to transport galactose was demonstrated by using galP disruption mutant strains of L. lactis MG1363. A galK deletion was constructed by replacement recombination, and the mutant strain was not able to ferment galactose. Disruption of the galE gene resulted in a deficiency in cell separation along with the appearance of a long-chain phenotype when cells were grown on glucose as the sole carbon source. Recovery of the wild-type phenotype for the galE mutant was obtained either by genetic complementation or by addition of galactose to the growth medium.

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TANG, MANSHU, ENOABASI ETOKIDEM, and KENT LAI. "The Leloir Pathway of Galactose Metabolism – A Novel Therapeutic Target for Hepatocellular Carcinoma." Anticancer Research 36, no. 12 (December 1, 2016): 6265–72. http://dx.doi.org/10.21873/anticanres.11221.

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Németh, Zoltán, László Kulcsár, Michel Flipphi, Anita Orosz, Maria Victoria Aguilar-Pontes, Ronald P. de Vries, Levente Karaffa, and Erzsébet Fekete. "l-Arabinose induces d-galactose catabolism via the Leloir pathway in Aspergillus nidulans." Fungal Genetics and Biology 123 (February 2019): 53–59. http://dx.doi.org/10.1016/j.fgb.2018.11.004.

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Bro, Christoffer, Steen Knudsen, Birgitte Regenberg, Lisbeth Olsson, and Jens Nielsen. "Improvement of Galactose Uptake in Saccharomyces cerevisiae through Overexpression of Phosphoglucomutase: Example of Transcript Analysis as a Tool in Inverse Metabolic Engineering." Applied and Environmental Microbiology 71, no. 11 (November 2005): 6465–72. http://dx.doi.org/10.1128/aem.71.11.6465-6472.2005.

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ABSTRACT Through genome-wide transcript analysis of a reference strain and two recombinant Saccharomyces cerevisiae strains with different rates of galactose uptake, we obtained information about the global transcriptional response to metabolic engineering of the GAL gene regulatory network. One of the recombinant strains overexpressed the gene encoding the transcriptional activator Gal4, and in the other strain the genes encoding Gal80, Gal6, and Mig1, which are negative regulators of the GAL system, were deleted. Even though the galactose uptake rates were significantly different in the three strains, we surprisingly did not find any significant changes in the expression of the genes encoding the enzymes catalyzing the first steps of the pathway (i.e., the genes encoding Gal2, Gal1, Gal7, and Gal10). We did, however, find that PGM2, encoding the major isoenzyme of phosphoglucomutase, was slightly up-regulated in the two recombinant strains with higher galactose uptake rates. This indicated that PGM2 is a target for overexpression in terms of increasing the flux through the Leloir pathway, and through overexpression of PGM2 the galactose uptake rate could be increased by 70% compared to that of the reference strain. Based on our findings, we concluded that phosphoglucomutase plays a key role in controlling the flux through the Leloir pathway, probably due to increased conversion of glucose-1-phosphate to glucose-6-phosphate. This conclusion was supported by measurements of sugar phosphates, which showed that there were increased concentrations of glucose-6-phosphate, galactose-6-phosphate, and fructose-6-phosphate in the strain construct overexpressing PGM2.

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Kikuchi, Atsuo, Yoichi Wada, Toshihiro Ohura, and Shigeo Kure. "The Discovery of GALM Deficiency (Type IV Galactosemia) and Newborn Screening System for Galactosemia in Japan." International Journal of Neonatal Screening 7, no. 4 (October 25, 2021): 68. http://dx.doi.org/10.3390/ijns7040068.

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The Leloir pathway, which consists of highly conserved enzymes, metabolizes galactose. Deficits in three enzymes in this pathway, namely galactose-1-phosphate uridylyltransferase (GALT), galactokinase (GALK1), and UDP-galactose-4′-epimerase (GALE), are associated with genetic galactosemia. We recently identified patients with galactosemia and biallelic variants in GALM, encoding galactose epimerase (GALM), an enzyme that is directly upstream of GALK1. GALM deficiency was subsequently designated as type IV galactosemia. Currently, all the published patients with biallelic GALM variants were found through newborn screening in Japan. Here, we review GALM deficiency and describe how we discovered this relatively mild but not rare disease through the newborn screening system in Japan.

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Aleksandrzak-Piekarczyk, Tamara, Jan Kok, Pierre Renault, and Jacek Bardowski. "Alternative Lactose Catabolic Pathway in Lactococcus lactis IL1403." Applied and Environmental Microbiology 71, no. 10 (October 2005): 6060–69. http://dx.doi.org/10.1128/aem.71.10.6060-6069.2005.

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ABSTRACT In this study, we present a glimpse of the diversity of Lactococcus lactis subsp. lactis IL1403 β-galactosidase phenotype-negative mutants isolated by negative selection on solid media containing cellobiose or lactose and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside), and we identify several genes essential for lactose assimilation. Among these are ccpA (encoding catabolite control protein A), bglS (encoding phospho-β-glucosidase), and several genes from the Leloir pathway gene cluster encoding proteins presumably essential for lactose metabolism. The functions of these genes were demonstrated by their disruption and testing of the growth of resultant mutants in lactose-containing media. By examining the ccpA and bglS mutants for phospho-β-galactosidase activity, we showed that expression of bglS is not under strong control of CcpA. Moreover, this analysis revealed that although BglS is homologous to a putative phospho-β-glucosidase, it also exhibits phospho-β-galactosidase activity and is the major enzyme in L. lactis IL1403 involved in lactose hydrolysis.

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Neves, Ana R., Wietske A. Pool, Ana Solopova, Jan Kok, Helena Santos, and Oscar P. Kuipers. "Towards Enhanced Galactose Utilization by Lactococcus lactis." Applied and Environmental Microbiology 76, no. 21 (September 17, 2010): 7048–60. http://dx.doi.org/10.1128/aem.01195-10.

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ABSTRACT Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

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Audy, Julie, Steve Labrie, Denis Roy, and Gisèle LaPointe. "Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002." Microbiology 156, no. 3 (March 1, 2010): 653–64. http://dx.doi.org/10.1099/mic.0.033720-0.

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The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080±120 mg EPS l−1 (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512±63, 564±165 and 616±93 mg EPS l−1, respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production (galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase (wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.

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Jónás, Ágota, Erzsébet Fekete, Zoltán Németh, Michel Flipphi, and Levente Karaffa. "D-galactose catabolism inPenicillium chrysogenum: Expression analysis of the structural genes of the Leloir pathway." Acta Biologica Hungarica 67, no. 3 (September 2016): 318–32. http://dx.doi.org/10.1556/018.67.2016.3.9.

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Seibel, Jürgen, Rafael Beine, Roxana Moraru, Carinna Behringer, and Klaus Buchholz. "A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases." Biocatalysis and Biotransformation 24, no. 1-2 (January 2006): 157–65. http://dx.doi.org/10.1080/10242420500538274.

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Ross, Kerry L., Charity N. Davis, and Judith L. Fridovich-Keil. "Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast." Molecular Genetics and Metabolism 83, no. 1-2 (September 2004): 103–16. http://dx.doi.org/10.1016/j.ymgme.2004.07.005.

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Taylor Fischer, S., Allison B. Frederick, ViLinh Tran, Shuzhao Li, Dean P. Jones, and Judith L. Fridovich‐Keil. "Metabolic perturbations in classic galactosemia beyond the Leloir pathway: Insights from an untargeted metabolomic study." Journal of Inherited Metabolic Disease 42, no. 2 (January 22, 2019): 254–63. http://dx.doi.org/10.1002/jimd.12007.

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Wang, Kuei-Chen, Syue-Yi Lyu, Yu-Chen Liu, Chin-Yuan Chang, Chang-Jer Wu, and Tsung-Lin Li. "Insights into the binding specificity and catalytic mechanism ofN-acetylhexosamine 1-phosphate kinases through multiple reaction complexes." Acta Crystallographica Section D Biological Crystallography 70, no. 5 (April 30, 2014): 1401–10. http://dx.doi.org/10.1107/s1399004714004209.

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Utilization ofN-acetylhexosamine in bifidobacteria requires the specific lacto-N-biose/galacto-N-biose pathway, a pathway differing from the Leloir pathway while establishing symbiosis between humans and bifidobacteria. The genelnpBin the pathway encodes a novel hexosamine kinase NahK, which catalyzes the formation ofN-acetylhexosamine 1-phosphate (GlcNAc-1P/GalNAc-1P). In this report, seven three-dimensional structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP from bothBifidobacterium longum(JCM1217) andB. infantis(ATCC15697) were solved at resolutions of 1.5–2.2 Å. NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The NahK structures presented here represent the first multiple reaction complexes of the enzyme. This structural information reveals the molecular basis for the recognition of the given substrates and products, GlcNAc/GalNAc, GlcNAc-1P/GalNAc-1P, ATP/ADP and Mg2+, and provides insights into the catalytic mechanism, enabling NahK and mutants thereof to form a choice of biocatalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.

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Bettenbrock, Katja, Ulrike Siebers, Petra Ehrenreich, and Carl-Alfred Alpert. "Lactobacillus casei 64H Contains a Phosphoenolpyruvate-Dependent Phosphotransferase System for Uptake of Galactose, as Confirmed by Analysis of ptsH and Differentgal Mutants." Journal of Bacteriology 181, no. 1 (January 1, 1999): 225–30. http://dx.doi.org/10.1128/jb.181.1.225-230.1999.

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ABSTRACT Galactose metabolism in Lactobacillus casei 64H was analyzed by genetic and biochemical methods. Mutants with defects inptsH, galK, or the tagatose 6-phosphate pathway were isolated either by positive selection using 2-deoxyglucose or 2-deoxygalactose or by an enrichment procedure with streptozotocin.ptsH mutations abolish growth on lactose, cellobiose,N-acetylglucosamine, mannose, fructose, mannitol, glucitol, and ribitol, while growth on galactose continues at a reduced rate. Growth on galactose is also reduced, but not abolished, ingalK mutants. A mutation in galK in combination with a mutation in the tagatose 6-phosphate pathway results in sensitivity to galactose and lactose, while a galK mutation in combination with a mutation in ptsH completely abolishes galactose metabolism. Transport assays, in vitro phosphorylation assays, and thin-layer chromatography of intermediates of galactose metabolism also indicate the functioning of a permease/Leloir pathway and a phosphoenolpyruvate-dependent phosphotransferase system (PTS)/tagatose 6-phosphate pathway. The galactose-PTS is induced by growth on either galactose or lactose, but the induction kinetics for the two substrates are different.

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Remy, E., M. Meyer, F. Blaise, U. K. Simon, D. Kuhn, M. H. Balesdent, and T. Rouxel. "A Key Enzyme of the Leloir Pathway Is Involved in Pathogenicity of Leptosphaeria maculans Toward Oilseed Rape." Molecular Plant-Microbe Interactions® 22, no. 6 (June 2009): 725–36. http://dx.doi.org/10.1094/mpmi-22-6-0725.

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Agrobacterium tumefaciens-mediated random insertional mutagenesis was used to investigate pathogenicity determinants in Leptosphaeria maculans. One tagged nonpathogenic mutant, termed m186, is analyzed in detail here. Microscopic analyses of infected plant tissues revealed that m186 is specifically blocked at the invasive growth phase after an unaffected initial penetration stage and is unable to switch to the necrotrophic lifestyle. In addition, m186 exhibits an altered cell wall and seems to be affected in its ability to produce cell-wall-degrading enzymes. The T-DNA insertion occurs in the intergenic region between two head-to-tail genes, leading to a constitutive upregulation of their expression. Complementation experiments showed that only one of these two genes, Lmepi, fully accounts for the mutant phenotype. Bioinformatics and expression analyses along with functional studies suggested that the Lmepi gene encodes for the highly conserved UDP-glucose-4-epimerase, a key enzyme of the Leloir pathway involved in galactose metabolism. For the third time, this study highlights the intimate connection between primary metabolism and pathogenicity in L. maculans. This finding, along with similar data obtained from the related species Stagonospora nodorum, indicates the importance of in planta nutrition for the success of infection of plants by fungi belonging to class Dothideomycete.

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Aleksandrzak‐Piekarczyk, Tamara, Katarzyna Szatraj, and Katarzyna Kosiorek. "GlaR (YugA)—a novel RpiR‐family transcription activator of the Leloir pathway of galactose utilization inLactococcus lactisIL1403." MicrobiologyOpen 8, no. 5 (August 11, 2018): e00714. http://dx.doi.org/10.1002/mbo3.714.

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Meyer, J., A. Walker-Jonah, and C. P. Hollenberg. "Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 11 (November 1991): 5454–61. http://dx.doi.org/10.1128/mcb.11.11.5454-5461.1991.

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We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

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Meyer, J., A. Walker-Jonah, and C. P. Hollenberg. "Galactokinase encoded by GAL1 is a bifunctional protein required for induction of the GAL genes in Kluyveromyces lactis and is able to suppress the gal3 phenotype in Saccharomyces cerevisiae." Molecular and Cellular Biology 11, no. 11 (November 1991): 5454–61. http://dx.doi.org/10.1128/mcb.11.11.5454.

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We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

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Anbukkarasi, Kaliyaperumal, Dhiraj Kumar Nanda, Thiyagamoorthy UmaMaheswari, Thiagarajan Hemalatha, Prashant Singh, and Rameshwar Singh. "Assessment of expression of Leloir pathway genes in wild-type galactose-fermenting Streptococcus thermophilus by real-time PCR." European Food Research and Technology 239, no. 5 (August 12, 2014): 895–903. http://dx.doi.org/10.1007/s00217-014-2286-9.

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Levander, Fredrik, and Peter Rådström. "Requirement for Phosphoglucomutase in Exopolysaccharide Biosynthesis in Glucose- and Lactose-Utilizing Streptococcus thermophilus." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2734–38. http://dx.doi.org/10.1128/aem.67.6.2734-2738.2001.

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ABSTRACT To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growingStreptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. ThepgmA gene, encoding PGM in S. thermophilusLY03, was identified and cloned. The gene was functional inEscherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.

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Reshma, S. V., Nitish Sathyanarayanan, and H. G. Nagendra. "Characterization of hypothetical protein VNG0128C fromHalobacteriumNRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism." Journal of Biomolecular Structure and Dynamics 33, no. 8 (November 14, 2014): 1743–55. http://dx.doi.org/10.1080/07391102.2014.969313.

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Frey, Perry A. "The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose." FASEB Journal 10, no. 4 (March 1996): 461–70. http://dx.doi.org/10.1096/fasebj.10.4.8647345.

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Afzal, Muhammad, Sulman Shafeeq, Irfan Manzoor, and Oscar P. Kuipers. "GalR Acts as a Transcriptional Activator of galKT in the Presence of Galactose in Streptococcus pneumoniae." Journal of Molecular Microbiology and Biotechnology 25, no. 6 (2015): 363–71. http://dx.doi.org/10.1159/000439429.

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We explored the regulatory mechanism of Leloir pathway genes in Streptococcus pneumoniae D39. Here, we demonstrate that the expression of galKT is galactose dependent. By microarray analysis and quantitative RT-PCR, we further show the role of the transcriptional regulator GalR, present upstream of galKT, as a transcriptional activator of galKT in the presence of galactose. Moreover, we predict a 19-bp regulatory site (5′-GATAGTTTAGTAAAATTTT-3′) for the transcriptional regulator GalR in the promoter region of galK, which is also highly conserved in other streptococci. Growth comparison of D39 ΔgalK with the D39 wild type grown in the presence of galactose shows that galK is required for the proper growth of S. pneumoniae on galactose.

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Nakamya, Mary, Moses Ayoola, Leslie Shack, Edwin Swiatlo, and Bindu Nanduri. "The Effect of Impaired Polyamine Transport on Pneumococcal Transcriptome." Pathogens 10, no. 10 (October 14, 2021): 1322. http://dx.doi.org/10.3390/pathogens10101322.

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Infections due to Streptococcus pneumoniae, a commensal in the nasopharynx, still claim a significant number of lives worldwide. Genome plasticity, antibiotic resistance, and limited serotype coverage of the available polysaccharide-based conjugate vaccines confounds therapeutic interventions to limit the spread of this pathogen. Pathogenic mechanisms that allow successful adaption and persistence in the host could be potential innovative therapeutic targets. Polyamines are ubiquitous polycationic molecules that regulate many cellular processes. We previously reported that deletion of polyamine transport operon potABCD, which encodes a putrescine/spermidine transporter (ΔpotABCD), resulted in an unencapsulated attenuated phenotype. Here, we characterize the transcriptome, metabolome, and stress responses of polyamine transport-deficient S. pneumoniae. Compared with the wild-type strain, the expression of genes involved in oxidative stress responses and the nucleotide sugar metabolism was reduced, while expression of genes involved in the Leloir, tagatose, and pentose phosphate pathways was higher in ΔpotABCD. A metabolic shift towards the pentose phosphate pathway will limit the synthesis of precursors of capsule polysaccharides. Metabolomics results show reduced levels of glutathione and pyruvate in the mutant. Our results also show that the potABCD operon protects pneumococci against hydrogen peroxide and nitrosative stress. Our findings demonstrate the importance of polyamine transport in pneumococcal physiology that could impact in vivo fitness. Thus, polyamine transport in pneumococci represents a novel target for therapeutic interventions.

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Boucher, Isabelle, Christian Vadeboncoeur, and Sylvain Moineau. "Characterization of Genes Involved in the Metabolism of α-Galactosides by Lactococcus raffinolactis." Applied and Environmental Microbiology 69, no. 7 (July 2003): 4049–56. http://dx.doi.org/10.1128/aem.69.7.4049-4056.2003.

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ABSTRACT Lactococcus raffinolactis, unlike most lactococci, is able to ferment α-galactosides, such as melibiose and raffinose. More than 12 kb of chromosomal DNA from L. raffinolactis ATCC 43920 was sequenced, including the α-galactosidase gene and genes involved in the Leloir pathway of galactose metabolism. These genes are organized into an operon containing aga (α-galactosidase), galK (galactokinase), and galT (galactose 1-phosphate uridylyltransferase). Northern blotting experiments revealed that this operon was induced by galactosides, such as lactose, melibiose, raffinose, and, to a lesser extent, galactose. Similarly, α-galactosidase activity was higher in lactose-, melibiose-, and raffinose-grown cells than in galactose-grown cells. No α-galactosidase activity was detected in glucose-grown cells. The expression of the aga-galKT operon was modulated by a regulator encoded by the upstream gene galR. The product of galR belongs to the LacI/GalR family of transcriptional regulators. In L. lactis, L. raffinolactis GalR acted as a repressor of aga and lowered the enzyme activity by more than 20-fold. We suggest that the expression of the aga operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides.

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McAuley, Megan, Helena Kristiansson, Meilan Huang, Angel L. Pey, and David J. Timson. "Galactokinase promiscuity: a question of flexibility?" Biochemical Society Transactions 44, no. 1 (February 9, 2016): 116–22. http://dx.doi.org/10.1042/bst20150188.

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Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering.

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Bhat, P. J., and J. E. Hopper. "The mechanism of inducer formation in gal3 mutants of the yeast galactose system is independent of normal galactose metabolism and mitochondrial respiratory function." Genetics 128, no. 2 (June 1, 1991): 233–39. http://dx.doi.org/10.1093/genetics/128.2.233.

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Abstract Saccharomyces cerevisiae cells defective in GAL3 function exhibit either one of two phenotypes. The gal3 mutation in an otherwise normal cell causes a 2-5-day delay in the galactose triggered induction of GAL/MEL gene transcription. This long term adaptation (LTA) phenotype has been ascribed to inefficient inducer formation. The gal3 mutation causes a noninducible phenotype for GAL/MEL transcription if cells are defective in Leloir pathway function, in glycolysis or in respiratory function. It was recently shown that multiple copies of the intact GAL1 gene partially suppress the LTA phenotype of gal3 cells. Here we report that constitutively expressed GAL1 restored gal3 mutants to the rapidly inducible phenotype characteristic of wild-type cells and conferred rapid inducibility to gal3 gal10, gal3 gal7 or gal3 rho- strains that are normally noninducible. As shown by immunoblot analysis, the GAL1-mediated induction exhibits phosphorylation of the GAL4 protein, suggesting a mechanism similar to GAL3-mediated induction. Altogether our results indicate that the deciding factor in the inducibility of the GAL/MEL genes in gal3 strains is the Gal3p-like activity of Gal1p. Based on the above we conclude that inducer formation does not require normal metabolism of galactose nor does it require mitochondrial respiratory function. These conclusions vitiate previous explanations for gal3 associated long-term adaptation and noninducible phenotypes.

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Sharma, Monica, Swati Sharma, Pallab Ray, and Anuradha Chakraborti. "Targeting Streptococcus pneumoniae UDP-glucose pyrophosphorylase (UGPase): in vitro validation of a putative inhibitor." Drug Target Insights 14, no. 1 (October 7, 2020): 26–33. http://dx.doi.org/10.33393/dti.2020.2103.

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Background: Genome plasticity of Streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide based vaccines. Therefore targets independent of capsular types are sought to control the pneumococcal pathogenicity. UcrDP-glucose pyrophosphorylase (UGPase) is one such desired candidate being responsible for the synthesis of UDP-glucose, a sugar-precursor in capsular biosynthesis and metabolic Leloir pathway. Being crucial to pneumococcal pathobiology, the effect of UGPase inhibition on virulence was evaluated in vitro. Methods: A putative inhibitor (UDP) was evaluated for effective inhibitory concentration in S. pneumoniae and A549 cells, its efficacy and toxicity. Effect of UDP on adherence and phagocytosis was measured in human respiratory epithelial (A549 and HEp-2) and macrophage (THP1 and J774.A.1) cell lines respectively. Results: A differential effective inhibitory concentration of UDP for UGPase inhibition was observed in S. pneumoniae and A549 cells i.e. 5 µM and 100 µM respectively. UDP treatments lowered percent cytotoxicity in pneumococcal infected monolayers and didn't exert adverse effects on viabilities. S. pneumoniae adherence to host cells was decreased significantly with UDP treatments. UDP induced the secretion of IL-1β, TNF-α, IL-6, and IL-8 and increased pneumococcal phagocytosis. Conclusion: Our study shows UDP mediated decrease in the virulence of S. pneumoniae and demonstrates UDP as an effective inhibitor of pneumococcal UGPase.

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ZINSSER, VERONIKA L., STEFFEN LINDERT, SAMANTHA BANFORD, ELIZABETH M. HOEY, ALAN TRUDGETT, and DAVID J. TIMSON. "UDP-galactose 4′-epimerase from the liver fluke, Fasciola hepatica: biochemical characterization of the enzyme and identification of inhibitors." Parasitology 142, no. 3 (August 15, 2014): 463–72. http://dx.doi.org/10.1017/s003118201400136x.

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SUMMARYThe Leloir pathway enzyme uridine diphosphate (UDP)-galactose 4′-epimerase from the common liver fluke Fasciola hepatica (FhGALE) was identified and characterized. The enzyme can be expressed in, and purified from, Escherichia coli. The recombinant enzyme is active: the Km (470 μm) is higher than the corresponding human enzyme (HsGALE), whereas the kcat (2·3 s−1) is substantially lower. FhGALE binds NAD+ and has shown to be dimeric by analytical gel filtration. Like the human and yeast GALEs, FhGALE is stabilized by the substrate UDP-galactose. Molecular modelling predicted that FhGALE adopts a similar overall fold to HsGALE and that tyrosine 155 is likely to be the catalytically critical residue in the active site. In silico screening of the National Cancer Institute Developmental Therapeutics Program library identified 40 potential inhibitors of FhGALE which were tested in vitro. Of these, 6 showed concentration-dependent inhibition of FhGALE, some with nanomolar IC50 values. Two inhibitors (5-fluoroorotate and N-[(benzyloxy)carbonyl]leucyltryptophan) demonstrated selectivity for FhGALE over HsGALE. These compounds also thermally destabilized FhGALE in a concentration-dependent manner. Interestingly, the selectivity of 5-fluoroorotate was not shown by orotic acid, which differs in structure by 1 fluorine atom. These results demonstrate that, despite the structural and biochemical similarities of FhGALE and HsGALE, it is possible to discover compounds which preferentially inhibit FhGALE.

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FORTINA, MARIA GRAZIA, GIOVANNI RICCI, and FRANCESCA BORGO. "A Study of Lactose Metabolism in Lactococcus garvieae Reveals a Genetic Marker for Distinguishing between Dairy and Fish Biotypes." Journal of Food Protection 72, no. 6 (June 1, 2009): 1248–54. http://dx.doi.org/10.4315/0362-028x-72.6.1248.

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Dairy and fish isolates of Lactococcus garvieae were tested for their ability to utilize lactose and to grow in milk. Fish isolates were unable to assimilate lactose, but unexpectedly, they possessed the ability to grow in milk. Genetic studies, carried out constructing different vectorette libraries, provided evidence that in fish isolates, no genes involved in lactose utilization were present. For L. garvieae dairy isolates, a single system for the catabolism of lactose was found. It consists of a lactose transport and hydrolysis depending on a phosphoenolpyruvate-dependent phosphotransferase system combined with a phospho-β-galactosidase. The genes involved were highly similar at the nucleotide sequence level to their counterparts in Lactococcus lactis; however, while in many L. lactis strains these genes are plasmid encoded, in L. garvieae they are chromosomally located. Thus, in the species L. garvieae, the phospho-β-galactosidase gene, detectable in all strains of dairy origin but lacking in fish isolates, can be considered a reliable genetic marker for distinguishing biotypes in the two diverse ecological niches. Moreover, we obtained information regarding the complete nucleotide sequence of the gal operon in L. garvieae, consisting of a galactose permease and the Leloir pathway enzymes. This is one of the first reports concerning the determination of the nucleotide sequences of genes (other than the 16S rDNA gene) in L. garvieae and should be considered a step in a continuous effort to explore the genome of this species, with the aim of determining the real relationship between the presence of L. garvieae in dairy products and food safety.

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Vaughan, Elaine E., Patrick T. C. van den Bogaard, Pasquale Catzeddu, Oscar P. Kuipers, and Willem M. de Vos. "Activation of Silent gal Genes in thelac-gal Regulon of Streptococcus thermophilus." Journal of Bacteriology 183, no. 4 (February 15, 2001): 1184–94. http://dx.doi.org/10.1128/jb.183.4.1184-1194.2001.

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ABSTRACT Streptococcus thermophilus strain CNRZ 302 is unable to ferment galactose, neither that generated intracellularly by lactose hydrolysis nor the free sugar. Nevertheless, sequence analysis and complementation studies with Escherichia coli demonstrated that strain CNRZ 302 contained structurally intact genes for the Leloir pathway enzymes. These were organized into an operon in the ordergalKTE, which was preceded by a divergently transcribed regulator gene, galR, and followed by a galMgene and the lactose operon lacSZ. Results of Northern blot analysis showed that the structural gal genes were transcribed weakly, and only in medium containing lactose, by strain CNRZ 302. However, in a spontaneous galactose-fermenting mutant, designated NZ302G, the galKTE genes were well expressed in cells grown on lactose or galactose. In both CNRZ 302 and the Gal+ mutant NZ302G, the transcription of thegalR gene was induced by growth on lactose. Disruption ofgalR indicated that it functioned as a transcriptional activator of both the gal and lac operons while negatively regulating its own expression. Sequence analysis of thegal promoter regions of NZ302G and nine other independently isolated Gal+ mutants of CNRZ 302 revealed mutations at three positions in the galK promoter region, which included substitutions at positions −9 and −15 as well as a single-base-pair insertion at position −37 with respect to the main transcription initiation point. Galactokinase activity measurements and analysis ofgusA reporter gene fusions in strains containing the mutated promoters suggested that they were gal promoter-up mutations. We propose that poor expression of the gal genes in the galactose-negative S. thermophilus CNRZ 302 is caused by naturally occurring mutations in the galKpromoter.

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Febres-Aldana, Christopher A., Liset Pelaez, Meredith S. Wright, Ossama M. Maher, Anthony J. Febres-Aldana, Jun Sasaki, Parul Jayakar, et al. "A Case of UDP-Galactose 4′-Epimerase Deficiency Associated with Dyshematopoiesis and Atrioventricular Valve Malformations: An Exceptional Clinical Phenotype Explained by Altered N-Glycosylation with Relative Preservation of the Leloir Pathway." Molecular Syndromology 11, no. 5-6 (2020): 320–30. http://dx.doi.org/10.1159/000511343.

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The generalized form of UDP-galactose-4′-epimerase (GALE) deficiency causes hypotonia, failure to thrive, cataracts, and liver failure. Individuals with non-generalized forms may remain asymptomatic with uncertain long-term outcomes. We report a 2-year-old child compound heterozygous for GALE p.R51W/p.G237D who never developed symptoms of classic galactosemia but has a history of congenital combined mitral and tricuspid valve malformation and pyloric stenosis, and presented with pancytopenia. Variant pathogenicity was supported by predictive computational tools and decreased GALE activity measured in erythrocytes. GALE function extends to the biosynthesis of glycans by epimerization of UDP-N-acetyl-galactosamine and -glucosamine. Interrogation of the Gene Ontology consortium database revealed several putative proteins involved in normal hematopoiesis and atrioventricular valve morphogenesis, requiring N-glycosylation for adequate functionality. We hypothesize that by limiting substrate supply due to GALE deficiency, alterations in N-linked protein glycosylation can explain the patient’s phenotype.

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Vaughan, Elaine E., R. David Pridmore, and Beat Mollet. "Transcriptional Regulation and Evolution of Lactose Genes in the Galactose-Lactose Operon of Lactococcus lactisNCDO2054." Journal of Bacteriology 180, no. 18 (September 15, 1998): 4893–902. http://dx.doi.org/10.1128/jb.180.18.4893-4902.1998.

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ABSTRACT The genetics of lactose utilization within the slow-lactose-fermenting Lactococcus lactis strain NCDO2054 was studied with respect to the organization, expression, and evolution of the lac genes. Initially the β-galactosidase gene (lacZ) was cloned by complementation of an Escherichia coli mutant on a 7-kb HpaI fragment. Nucleotide sequence analysis of the complete fragment revealed part of a gal-lac operon, and the genes were characterized by inactivation and complementation analyses and in vitro enzyme activity measurements. The gene order isgalK-galT-lacA-lacZ-galE; the gal genes encode enzymes of the Leloir pathway for galactose metabolism, andlacA encodes a galactoside acetyltransferase. ThegalT and galE genes of L. lactisLM0230 (a lactose plasmid-cured derivative of the fast-lactose-fermenting L. lactis C2) were highly similar at the nucleotide sequence level to their counterparts in strain NCDO2054 and, furthermore, had the same gene order except for the presence of the intervening lacA-lacZ strain NCDO2054. Analysis of mRNA for the gal and lac genes revealed an unusual transcriptional organization for the operon, with a surprisingly large number of transcriptional units. The regulation of the lac genes was further investigated by using fusions consisting of putative promoter fragments and the promoterless β-glucuronidase gene (gusA) from E. coli, which identified three lactose-inducible intergenic promoters in the gal-lac operon. The greater similarity of thelacA and lacZ genes to homologs in gram-negative organisms than to those of gram-positive bacteria, in contrast to the homologies of the gal genes, suggests that the genes within the gal operon of L. lactisNCDO2054 have been recently acquired. Thus, thelacA-lacZ genes appear to have engaged the promoters of thegal operon in order to direct and control their expression.

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Vaillancourt, Katy, Jean-Dominique LeMay, Maryse Lamoureux, Michel Frenette, Sylvain Moineau, and Christian Vadeboncoeur. "Characterization of a Galactokinase-Positive Recombinant Strain of Streptococcus thermophilus." Applied and Environmental Microbiology 70, no. 8 (August 2004): 4596–603. http://dx.doi.org/10.1128/aem.70.8.4596-4603.2004.

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ABSTRACT The lactic acid bacterium Streptococcus thermophilus is widely used by the dairy industry for its ability to transform lactose, the primary sugar found in milk, into lactic acid. Unlike the phylogenetically related species Streptococcus salivarius, S. thermophilus is unable to metabolize and grow on galactose and thus releases substantial amounts of this hexose into the external medium during growth on lactose. This metabolic property may result from the inability of S. thermophilus to synthesize galactokinase, an enzyme of the Leloir pathway that phosphorylates intracellular galactose to generate galactose-1-phosphate. In this work, we report the complementation of Gal− strain S. thermophilus SMQ-301 with S. salivarius galK, the gene that codes for galactokinase, and the characterization of recombinant strain SMQ-301K01. The recombinant strain, which was obtained by transformation of strain SMQ-301 with pTRKL2TK, a plasmid bearing S. salivarius galK, grew on galactose with a generation time of 55 min, which was almost double the generation time on lactose. Data confirmed that (i) the ability of SMQ-301K01 to grow on galactose resulted from the expression of S. salivarius galK and (ii) transcription of the plasmid-borne galK gene did not require GalR, a transcriptional regulator of the gal and lac operons, and did not interfere with the transcription of these operons. Unexpectedly, recombinant strain SMQ-301K01 still expelled galactose during growth on lactose, but only when the amount of the disaccharide in the medium exceeded 0.05%. Thus, unlike S. salivarius, the ability to metabolize galactose was not sufficient for S. thermophilus to simultaneously metabolize the glucose and galactose moieties of lactose. Nevertheless, during growth in milk and under time-temperature conditions that simulated those used to produce mozzarella cheese, the recombinant Gal+ strain grew and produced acid more rapidly than the Gal− wild-type strain.

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Figueroa, Carlos M., John E. Lunn, and Alberto A. Iglesias. "Nucleotide-sugar metabolism in plants: the legacy of Luis F. Leloir." Journal of Experimental Botany 72, no. 11 (May 5, 2021): 4053–67. http://dx.doi.org/10.1093/jxb/erab109.

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Abstract This review commemorates the 50th anniversary of the Nobel Prize in Chemistry awarded to Luis F. Leloir ‘for his discovery of sugar-nucleotides and their role in the biosynthesis of carbohydrates’. He and his co-workers discovered that activated forms of simple sugars, such as UDP-glucose and UDP-galactose, are essential intermediates in the interconversion of sugars. They elucidated the biosynthetic pathways for sucrose and starch, which are the major end-products of photosynthesis, and for trehalose. Trehalose 6-phosphate, the intermediate of trehalose biosynthesis that they discovered, is now a molecule of great interest due to its function as a sugar signalling metabolite that regulates many aspects of plant metabolism and development. The work of the Leloir group also opened the doors to an understanding of the biosynthesis of cellulose and other structural cell wall polysaccharides (hemicelluloses and pectins), and ascorbic acid (vitamin C). Nucleotide-sugars also serve as sugar donors for a myriad of glycosyltransferases that conjugate sugars to other molecules, including lipids, phytohormones, secondary metabolites, and proteins, thereby modifying their biological activity. In this review, we highlight the diversity of nucleotide-sugars and their functions in plants, in recognition of Leloir’s rich and enduring legacy to plant science.

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HUTKINS, R. W., and H. A. MORRIS. "Carbohydrate Metabolism by Streptococcus thermophilus: A Review." Journal of Food Protection 50, no. 10 (October 1, 1987): 876–84. http://dx.doi.org/10.4315/0362-028x-50.10.876.

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Despite the widespread use of Streptococcus thermophilus as a starter culture in the manufacture of many fermented dairy products, only recently has an understanding of the basic processes regarding carbohydrate metabolism been developed. Although S. thermophilus is related to other lactic streptococci by virtue of their common use in dairy fermentations, available information indicates that S. thermophilus is serologically, genetically, and physiologically distinct from the Group N, mesophilic streptococci. Carbohydrate metabolism, in particular, occurs by different processes in S. thermophilus than in the Group N streptococci (Streptococcus lactis and Streptococcus cremoris). The latter organisms utilize lactose by a specific phosphoenolpyruvate-dependent phosphotransferase system in which the lactose hydrolysis products, glucose and galactyose-6-phosphate, are concurrently metabolized to lactic acid. In contrast, S. thermophilus lacks phosphotransferase activity and instead possesses a lactose permease. After hydrolysis by β-galactosidase, only glucose is further metabolized and galactose is released into the extracellular medium. Most strains are unable to ferment galactose and are phenotypically galactose-negative. The rapid growth rates of S. thermophilus on lactose and slow growth rates on glucose and galactose are likely due to the differences between the lactose and monosaccharide transport activities. Galactose transport by S. thermophilus requires an exogenous energy source and is mediated by a galactose permease. Galactose is further metabolized in galactose-positive cells by the enzymes of the Leloir pathway, specifically, galactokinase, galactose-1-phosphate uridyl transferase, and uridine-5-diphospho-glucose-4-epimerase. The latter two enzymes are eonstituitively expressed; however, in galactose-positive cells galactokinase and the galactose permease are induced by galactose in the absence of lactose. The phenotypic differences between galactose-positive and galactose-negative S. thermophilus are, in part, due to differences in the galactokinase and galactose permease activities. Galactose released into the medium by lactose-grown, galactose-positive cells can be subsequently metabolized, homofermentatively, to lactic acid. However, the important practical implications of released galactose has produced the need for isolation and development of S. thermophilus strains which ferment the lactose components, glucose and galactose, completely and simultaneously.

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49

Lelono, Eko Budi. "The Migration Pathway Of Some Selected Australian Palynomorphs From Their Origin To Se Asia." Scientific Contributions Oil and Gas 35, no. 2 (March 10, 2022): 49–56. http://dx.doi.org/10.29017/scog.35.2.777.

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This paper proposes the alternative dispersal route of Australian elements of Dacrydium and Casuarina from their origin to Southeast Asia. It was previously thought that these Australian affi nities migrated to Sunda region following the collision of the Australian and the Asian plates at the Oligo-Miocene boundary (Morley, 1998 and 2000). The subsequent study by Lelono (2007) extended the record of these two taxa from the Oligo-Miocene boundary to the base Oligocene. This is unlikely, since at the time of basal Oligocene, when these pollen types fi rst appear, the Australian land mass would have been some 1000 kms south of the East Java area. Therefore, this fact led Lelono (2007) to propose the earlier arrival of the Gondwanan fragment to this area in Early Oligocene. However, recent records of Dacrydium have been reported from the Early Eocene of the Ninety East Ridge (55 Ma) and the Indian subcontinent (50 Ma) (Morley, 2009). This implies to the alternative dispersal route of this pollen. It is possible that Dacrydium dispersed into SE Asia prior to the Early Oligocene via the Ninety East Ridge and the Indian plate, and subsequently its distribution across the Sunda region and Indochina was limited by palaeoclimate, explaining why it is present in some areas of the Sunda region, but not others. Mean while, a model to explain the dispersal of Casuarina remains unresolved, since migration via India is unlikely as there is no pollen record from the Indian subcontinent. Therefore, long distance dispersal may be a possibility for this pollen.

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50

Zeng, Lin, Satarupa Das, and Robert A. Burne. "Utilization of Lactose and Galactose by Streptococcus mutans: Transport, Toxicity, and Carbon Catabolite Repression." Journal of Bacteriology 192, no. 9 (February 26, 2010): 2434–44. http://dx.doi.org/10.1128/jb.01624-09.

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ABSTRACT 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 PlacA -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, EIIMan and EIILac, respectively, were shown to be the only effective transporters of galactose in S. mutans. Furthermore, disruption of manL, encoding EIIABMan, 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.

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Journal articles on the topic 'Leloir pathway'

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