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Andrew J Mccammon - One of the best experts on this subject based on the ideXlab platform.
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Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness
2015Co-Authors: Jacob D Durrant, Michael D Urbaniak, Michael A J Ferguson, Andrew J MccammonAbstract: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
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computer aided identification of trypanosoma brucei uridine diphosphate galactose 4 epimerase inhibitors toward the development of novel therapies for african sleeping sickness
Journal of Medicinal Chemistry, 2010Co-Authors: Jacob D Durrant, Michael D Urbaniak, Michael A J Ferguson, Andrew J MccammonAbstract: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 microM, 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.
Zbigniew Lazar - One of the best experts on this subject based on the ideXlab platform.
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Awakening the endogenous Leloir Pathway for efficient galactose utilization by Yarrowia lipolytica
Biotechnology for Biofuels, 2015Co-Authors: Zbigniew Lazar, Anne-Marie Crutz- Coq, Heber Gamboa-Meléndez, Cecile Neuveglise, Jean-marc NicaudAbstract:BackgroundProduction of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins.ResultsYarrowia lipolytica was found to express all the Leloir Pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir Pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose.ConclusionsThe construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.
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awakening the endogenous Leloir Pathway for efficient galactose utilization by yarrowia lipolytica
Biotechnology for Biofuels, 2015Co-Authors: Zbigniew Lazar, Cecile Neuveglise, Heber Gamboamelendez, Annemarie Crutzle Coq, Jean-marc NicaudAbstract:Production of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins. Yarrowia lipolytica was found to express all the Leloir Pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir Pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose. The construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.
Levente Karaffa - One of the best experts on this subject based on the ideXlab platform.
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d galactose catabolism in penicillium chrysogenum expression analysis of the structural genes of the Leloir Pathway
Acta Biologica Hungarica, 2016Co-Authors: Agota Jonas, Erzsebet Fekete, Zoltan Nemeth, Michel Flipphi, Levente KaraffaAbstract:In this study, we analyzed the expression of the structural genes encoding the five enzymes comprising the Leloir Pathway of D-galactose catabolism in the industrial cell factory Penicillium chrysogenum on various carbon sources. The genome of P. chrysogenum contains a putative galactokinase gene at the annotated locus Pc13g10140, the product of which shows strong structural similarity to yeast galactokinase that was expressed on lactose and D-galactose only. The expression profile of the galactose-1-phosphate uridylyl transferase gene at annotated locus Pc15g00140 was essentially similar to that of galactokinase. This is in contrast to the results from other fungi such as Aspergillus nidulans, Trichoderma reesei and A. niger, where the ortholog galactokinase and galactose-1-phosphate uridylyl transferase genes were constitutively expressed. As for the UDP-galactose-4-epimerase encoding gene, five candidates were identified. We could not detect Pc16g12790, Pc21g12170 and Pc20g06140 expression on any of th...
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d-Galactose uptake is nonfunctional in the conidiospores of Aspergillus niger
FEMS microbiology letters, 2012Co-Authors: Erzsebet Fekete, Erzsebet Sandor, Bernhard Seiboth, Christian P Kubicek, Ronald P. De Vries, Patricia A. Vankuyk, Éva Fekete, Benjamin Metz, Levente KaraffaAbstract:The majority of black Aspergilli (Aspergillus section Nigri), including Aspergillus niger, as well as many other Ascomycetes fail to germinate on d-galactose as a sole carbon source. Here, we provide evidence that the ability of A. niger to transport d-galactose is growth stage dependent, being absent in the conidiospores but present in the mycelia. Despite earlier claims, we could identify galactokinase activity in growing cells and all genes of the Leloir Pathway (responsible for channelling d-galactose into the EMP Pathway) are well induced on d-galactose (and also on lactose, d-xylose and l-arabinose) in the mycelial stage. Expression of all Leloir Pathway genes was also detectable in conidiospores, although galE (encoding a galactokinase) and galD (encoding a galactose-1-phosphate uridylyl transferase) were expressed poorly. These results suggest that the d-galactose-negative phenotype of A. niger conidiospores may be due to the lack of inducer uptake. Keywords: Aspergillus niger; d-galactose; Leloir Pathway; uptake; germination
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lack of aldose 1 epimerase in hypocrea jecorina anamorph trichoderma reesei a key to cellulase gene expression on lactose
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Erzsebet Fekete, Bernhard Seiboth, Attila Szentirmai, Christian P Kubicek, Levente KaraffaAbstract:The heterodisaccharide lactose (1,4-O-beta-D-galactopyranosyl-D-glucose) induces cellulase formation in the ascomycete Hypocrea jecorina (= Trichoderma reesei). Lactose assimilation is slow, and the assimilation of its beta-D-galactose moiety depends mainly on the operation of a recently described reductive Pathway and depends less on the Leloir Pathway, which accepts only alpha-D-galactose. We therefore reasoned whether galactomutarotase [aldose 1-epimerase (AEP)] activity might limit lactose assimilation and thus influence cellulase formation. We identified three putative AEP-encoding genes (aep1, aep2, aep3) in H. jecorina, of which two encoded intracellular protein (AEP1 and AEP2) and one encoded an extracellular protein (AEP3). Although all three were transcribed, only the aep3 transcript was detected on lactose. However, no mutarotase activity was detected in the mycelia, their cell walls, or the extracellular medium during growth on lactose. Therefore, the effect of galactomutarotase activity on lactose assimilation was studied with H. jecorina strains expressing the C-terminal galactose mutarotase part of the Saccharomyces cerevisiae Gal10. These strains showed increased growth on lactose in a gene copy number-dependent manner, although their formation of extracellular beta-galactosidase activity and transcription of the genes encoding the first steps in the Leloir and the reductive Pathway was similar to the parental strain QM9414. Cellulase gene transcription on lactose dramatically decreased in these strains, but remained unaffected during growth on cellulose. Our data show that cellulase induction in H. jecorina by lactose requires the beta-anomer of D-galactose and reveal the lack of mutarotase activity during growth on lactose as an important key for cellulase formation on this sugar.
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induction of extracellular β galactosidase bga1 formation by d galactose in hypocrea jecorina is mediated by galactitol
Microbiology, 2007Co-Authors: Erzsebet Fekete, Levente Karaffa, Attila Szentirmai, Christian P Kubicek, Bernhard SeibothAbstract:The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 β-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of β-galactosidase at comparable growth rates. To know more about the actual inducer for β-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading Pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir Pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second Pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.
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the alternative d galactose degrading Pathway of aspergillus nidulans proceeds via l sorbose
Archives of Microbiology, 2004Co-Authors: Erzsebet Fekete, Levente Karaffa, Erzsebet Sandor, Bernhard Seiboth, Gyongyi Gyemant, Adel Sepsi, Attila Szentirmai, Istvan Banyai, Christian P KubicekAbstract:The catabolism of d-galactose in yeast depends on the enzymes of the Leloir Pathway. In contrast, Aspergillus nidulans mutants in galactokinase (galE) can still grow on d-galactose in the presence of ammonium—but not nitrate—ions as nitrogen source. A. nidulans galE mutants transiently accumulate high (400 mM) intracellular concentrations of galactitol, indicating that the alternative d-galactose degrading Pathway may proceed via this intermediate. The enzyme degrading galactitol was identified as l-arabitol dehydrogenase, because an A. nidulans loss-of-function mutant in this enzyme (araA1) did not show NAD+-dependent galactitol dehydrogenase activity, still accumulated galactitol but was unable to catabolize it thereafter, and a double galE/araA1 mutant was unable to grow on d-galactose or galactitol. The product of galactitol oxidation was identified as l-sorbose, which is a substrate for hexokinase, as evidenced by a loss of l-sorbose phosphorylating activity in an A. nidulans hexokinase (frA1) mutant. l-Sorbose catabolism involves a hexokinase step, indicated by the inability of the frA1 mutant to grow on galactitol or l-sorbose, and by the fact that a galE/frA1 double mutant of A. nidulans was unable to grow on d-galactose. The results therefore provide evidence for an alternative Pathway of d-galactose catabolism in A. nidulans that involves reduction of the d-galactose to galactitol and NAD+-dependent oxidation of galactitol by l-arabitol dehydrogenase to l-sorbose.
Jacob D Durrant - One of the best experts on this subject based on the ideXlab platform.
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Computer-Aided Identification of Trypanosoma brucei Uridine Diphosphate Galactose 4′-Epimerase Inhibitors: Toward the Development of Novel Therapies for African Sleeping Sickness
2015Co-Authors: Jacob D Durrant, Michael D Urbaniak, Michael A J Ferguson, Andrew J MccammonAbstract: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
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computer aided identification of trypanosoma brucei uridine diphosphate galactose 4 epimerase inhibitors toward the development of novel therapies for african sleeping sickness
Journal of Medicinal Chemistry, 2010Co-Authors: Jacob D Durrant, Michael D Urbaniak, Michael A J Ferguson, Andrew J MccammonAbstract: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 microM, 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.
Jean-marc Nicaud - One of the best experts on this subject based on the ideXlab platform.
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Awakening the endogenous Leloir Pathway for efficient galactose utilization by Yarrowia lipolytica
Biotechnology for Biofuels, 2015Co-Authors: Zbigniew Lazar, Anne-Marie Crutz- Coq, Heber Gamboa-Meléndez, Cecile Neuveglise, Jean-marc NicaudAbstract:BackgroundProduction of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins.ResultsYarrowia lipolytica was found to express all the Leloir Pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir Pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose.ConclusionsThe construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.
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awakening the endogenous Leloir Pathway for efficient galactose utilization by yarrowia lipolytica
Biotechnology for Biofuels, 2015Co-Authors: Zbigniew Lazar, Cecile Neuveglise, Heber Gamboamelendez, Annemarie Crutzle Coq, Jean-marc NicaudAbstract:Production of valuable metabolites by Yarrowia lipolytica using renewable raw materials is of major interest for sustainable food and energy. Galactose is a monosaccharide found in galactomannans, hemicelluloses, gums, and pectins. Yarrowia lipolytica was found to express all the Leloir Pathway genes for galactose utilization, which encode fully functional proteins. Gene organization and regulation in Y. lipolytica resembles filamentous fungi rather than Saccharomyces cerevisiae. After Y. lipolytica was grown on mixture of glucose and galactose, it was then able to metabolize galactose, including when glucose concentrations were higher than 4 g/L. However, glucose was still the preferred carbon source. Nonetheless, a strain overexpressing the four ylGAL genes of the Leloir Pathway was able to efficiently use galactose as its sole carbon source. This mutant was used to produce citric acid and lipids from galactose; the yields were comparable to or greater than that obtained for the parental strain (W29) on glucose. The construction of a Y. lipolytica strain able to produce citric acid and lipids from galactose is a very important step in bypassing issues related to the use of food-based substrates in industrial applications.