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Stephen W. Ragsdale - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of a methyltetrahydrofolate and corrinoid dependent Methyltransferase
Structure, 2000Co-Authors: Tzanko Doukov, Javier Seravalli, John J Stezowski, Stephen W. RagsdaleAbstract:Abstract Background: Methyltetrahydrofolate, corrinoid iron–sulfur Protein Methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation. It transfers the N 5 -methyl group from methyltetrahydrofolate (CH 3 -H 4 folate) to a cob(I)amide center in another Protein, the corrinoid iron–sulfur Protein. MeTr is a member of a family of Proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or-sarcino)pterin. We report the first structure of a Protein in this family. Results: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 A resolution using multiwavelength anomalous diffraction methods. The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold. The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity. This homology permitted the methyl-H 4 folate binding site to be modeled. The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the Methyltransferases and dihydropteroate synthetases. The most significant structural difference between these enzymes is in a loop structure above the active site. It is quite open in MeTr, where it can be modeled as the cobalamin binding site. Conclusions: The MeTr structure consists of a TIM barrel that embeds methyl-H 4 folate and cobamide. All related Methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site. The observed structure is consistent with either a ‘front' (N 5 ) or ‘back' (C 8a ) side protonation of CH 3 -H 4 folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).
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mechanism of transfer of the methyl group from 6s methyltetrahydrofolate to the corrinoid iron sulfur Protein catalyzed by the Methyltransferase from clostridium thermoaceticum a key step in the wood ljungdahl pathway of acetyl coa synthesis
Biochemistry, 1999Co-Authors: Javier Seravalli, Shaying Zhao, Stephen W. RagsdaleAbstract:The methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur Protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.
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a conformational change in the Methyltransferase from clostridium thermoaceticum facilitates the methyl transfer from 6s methyltetrahydrofolate to the corrinoid iron sulfur Protein in the acetyl coa pathway
Biochemistry, 1996Co-Authors: Shaying Zhao, Stephen W. RagsdaleAbstract:The methyltetrahydrofolate:corrinoid/iron−sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron−sulfur Protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate). This is an important reaction in the reductive acetyl-CoA pathway. The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the Protein [Zhao, S., Roberts, D. L., & Ragsdale, S. W. (1995) Biochemistry 34, 15075−15083]. In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change. As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pKa = 5.4) and the emission intensity increased (pKa = 5.1). The extrinsic fluorescence probe, 4,4‘-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change. The bis-ANS fluorescence...
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mechanistic studies of the Methyltransferase from clostridium thermoaceticum origin of the ph dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid iron sulfur Protein
Biochemistry, 1995Co-Authors: Shaying Zhao, D L Roberts, Stephen W. RagsdaleAbstract:A methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur Protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)
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the reductive acetyl coenzyme a pathway sequence and heterologous expression of active methyltetrahydrofolate corrinoid iron sulfur Protein Methyltransferase from clostridium thermoaceticum
Journal of Bacteriology, 1994Co-Authors: D L Roberts, Shaying Zhao, T Doukov, Stephen W. RagsdaleAbstract:The Methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur Protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell Protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains.
Shaying Zhao - One of the best experts on this subject based on the ideXlab platform.
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mechanism of transfer of the methyl group from 6s methyltetrahydrofolate to the corrinoid iron sulfur Protein catalyzed by the Methyltransferase from clostridium thermoaceticum a key step in the wood ljungdahl pathway of acetyl coa synthesis
Biochemistry, 1999Co-Authors: Javier Seravalli, Shaying Zhao, Stephen W. RagsdaleAbstract:The methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur Protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.
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a conformational change in the Methyltransferase from clostridium thermoaceticum facilitates the methyl transfer from 6s methyltetrahydrofolate to the corrinoid iron sulfur Protein in the acetyl coa pathway
Biochemistry, 1996Co-Authors: Shaying Zhao, Stephen W. RagsdaleAbstract:The methyltetrahydrofolate:corrinoid/iron−sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the methylation of a corrinoid/iron−sulfur Protein (C/Fe-SP) by the N5 methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate). This is an important reaction in the reductive acetyl-CoA pathway. The forward and reverse reactions of MeTr have a pH dependence that appears to reflect protonation of a group on the Protein [Zhao, S., Roberts, D. L., & Ragsdale, S. W. (1995) Biochemistry 34, 15075−15083]. In the work reported here, fluorescence and rapid reaction kinetics were used to demonstrate that this protonation elicits a rate-limiting conformational change. As the pH was lowered, the emission maximum for intrinsic tryptophan fluorescence underwent a red shift (pKa = 5.4) and the emission intensity increased (pKa = 5.1). The extrinsic fluorescence probe, 4,4‘-bis-1-phenylamino-8-napthalenesulfonate (bis-ANS) was used to report on the conformational change. The bis-ANS fluorescence...
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mechanistic studies of the Methyltransferase from clostridium thermoaceticum origin of the ph dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid iron sulfur Protein
Biochemistry, 1995Co-Authors: Shaying Zhao, D L Roberts, Stephen W. RagsdaleAbstract:A methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur Protein (C/Fe-SP). The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation. The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics. This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why [14C]methylcobyric acid was found as a product of labeling C. thermoaceticum cells with 14CO2 [Ljungdahl, L. G., Irion, E., & Wood, H. G. (1965) Biochemistry 4, 2771-2780]. Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III). The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5). Similar pH profiles were obtained by steady-state kinetics. The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3). A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS)
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the reductive acetyl coenzyme a pathway sequence and heterologous expression of active methyltetrahydrofolate corrinoid iron sulfur Protein Methyltransferase from clostridium thermoaceticum
Journal of Bacteriology, 1994Co-Authors: D L Roberts, Shaying Zhao, T Doukov, Stephen W. RagsdaleAbstract:The Methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur Protein in the acetyl coenzyme A pathway. MeTr was purified to homogeneity and shown to lack metals. The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell Protein. Regions in the sequence of MeTr and the E. coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains.
Jill E. Gready - One of the best experts on this subject based on the ideXlab platform.
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Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr): protonation state of the ligand and active-site residues.
The Journal of Physical Chemistry B, 2009Co-Authors: Hernan Alonso, Peter L. Cummins, Jill E. GreadyAbstract:Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH(3)THF) to the cobalt center of a corrinoid/iron-sulfur Protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH(3)THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK(a) increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH(3)THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK(a) values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the Protein environment was then considered. Different protonation states of CH(3)THF and active-site aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH(3)THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK(a) of N5 by about 3 units, leading to proton uptake upon CH(3)THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.
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methyltetrahydrofolate corrinoid iron sulfur Protein Methyltransferase metr protonation state of the ligand and active site residues
Journal of Physical Chemistry B, 2009Co-Authors: Hernan Alonso, Peter L. Cummins, Jill E. GreadyAbstract:Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH(3)THF) to the cobalt center of a corrinoid/iron-sulfur Protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH(3)THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK(a) increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH(3)THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK(a) values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the Protein environment was then considered. Different protonation states of CH(3)THF and active-site aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH(3)THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK(a) of N5 by about 3 units, leading to proton uptake upon CH(3)THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.
Christian P. Kubicek - One of the best experts on this subject based on the ideXlab platform.
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The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene
2016Co-Authors: Razieh Karimi Aghcheh, Irina S. Druzhinina, Lea Atanasova, Melinda Paholcsek, Benigno Aquino, Levente Karaffa, Christian P. KubicekAbstract:Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the Protein Methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1-LaeA- is part of the VELVET Protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression
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the velvet a orthologue vel1 of trichoderma reesei regulates fungal development and is essential for cellulase gene expression
PLOS ONE, 2014Co-Authors: Razieh Karimi Aghcheh, Irina S. Druzhinina, Lea Atanasova, Melinda Paholcsek, Benigno Aquino, Levente Karaffa, Zoltan Nemeth, Erzsebet Fekete, Erzsebet Sandor, Christian P. KubicekAbstract:Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the Protein Methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET Protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.
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The putative Protein Methyltransferase LAE1 of Trichoderma atroviride is a key regulator of asexual development and mycoparasitism.
PLoS ONE, 2013Co-Authors: Razieh Karimi Aghcheh, Irina S. Druzhinina, Christian P. KubicekAbstract:In Ascomycota the Protein Methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a Δlae1 and 30–50% increased in lae1-overexpressing (OElae1) strains. In darkness, Δlae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the Δlae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 s-glucanases, polyketide synthases and small cystein-rich secreted Proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.
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The putative Protein Methyltransferase LAE1 controls cellulase gene expression in Trichoderma reesei
Molecular Microbiology, 2012Co-Authors: Bernhard Seiboth, Razieh Karimi, Pallavi A. Phatale, Rita Linke, Lukas Hartl, Dominik G. Sauer, Kristina M. Smith, Scott E. Baker, Michael Freitag, Christian P. KubicekAbstract:Trichoderma reesei is an industrial producer of enzymes that degrade lignocellulosic polysaccharides to soluble monomers, which can be fermented to biofuels. Here we show that the expression of genes for lignocellulose degradation are controlled by the orthologous T. reesei Protein Methyltransferase LAE1. In a lae1 deletion mutant we observed a complete loss of expression of all seven cellulases, auxiliary factors for cellulose degradation, β-glucosidases and xylanases were no longer expressed. Conversely, enhanced expression of lae1 resulted in significantly increased cellulase gene transcription. Lae1-modulated cellulase gene expression was dependent on the function of the general cellulase regulator XYR1, but also xyr1 expression was LAE1-dependent. LAE1 was also essential for conidiation of T. reesei. Chromatin immunoprecipitation followed by high-throughput sequencing (‘ChIP-seq’) showed that lae1 expression was not obviously correlated with H3K4 di- or trimethylation (indicative of active transcription) or H3K9 trimethylation (typical for heterochromatin regions) in CAZyme coding regions, suggesting that LAE1 does not affect CAZyme gene expression by directly modulating H3K4 or H3K9 methylation. Our data demonstrate that the putative Protein Methyltransferase LAE1 is essential for cellulase gene expression in T. reesei through mechanisms that remain to be identified.
Hernan Alonso - One of the best experts on this subject based on the ideXlab platform.
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Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr): protonation state of the ligand and active-site residues.
The Journal of Physical Chemistry B, 2009Co-Authors: Hernan Alonso, Peter L. Cummins, Jill E. GreadyAbstract:Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH(3)THF) to the cobalt center of a corrinoid/iron-sulfur Protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH(3)THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK(a) increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH(3)THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK(a) values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the Protein environment was then considered. Different protonation states of CH(3)THF and active-site aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH(3)THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK(a) of N5 by about 3 units, leading to proton uptake upon CH(3)THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.
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methyltetrahydrofolate corrinoid iron sulfur Protein Methyltransferase metr protonation state of the ligand and active site residues
Journal of Physical Chemistry B, 2009Co-Authors: Hernan Alonso, Peter L. Cummins, Jill E. GreadyAbstract:Methyltetrahydrofolate:corrinoid/iron-sulfur Protein Methyltransferase (MeTr) catalyzes the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH(3)THF) to the cobalt center of a corrinoid/iron-sulfur Protein, a reaction similar to that of cobalamin-dependent methionine synthase (MetH). For such a reaction to occur, CH(3)THF is expected to be activated by a stereospecific protonation at the N5 position. It has been shown experimentally that binding to MeTr is associated with a pK(a) increase and proton uptake. The enzyme could achieve this by binding the unprotonated form of CH(3)THF, followed by specific protonation at the correct orientation. Here we have used computational approaches to investigate the protonation state of the ligand and active-site residues in MeTr. First, quantum mechanical (QM) methods with the PCM solvation model were used to predict protonation positions and pK(a) values of pterin, folate, and their analogues in an aqueous environment. After a reliable calibration of computational and experimental results was obtained, the effect of the Protein environment was then considered. Different protonation states of CH(3)THF and active-site aspartic residues (D75 and D160) were investigated using QM calculations of active-site fragment complexes and the perturbed quantum atom (PQA) approach within QM/MM simulations. The final free energy results indicate that the N5 position of the tetrahydropterin ring is the preferred protonation position of CH(3)THF when bound to the active site of MeTr, followed by Asp160. We also found that the active-site environment is likely to increase the pK(a) of N5 by about 3 units, leading to proton uptake upon CH(3)THF binding, as observed experimentally for MeTr. Some implications of the results are discussed for the MetH mechanism.
