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Xianghong Wang - One of the best experts on this subject based on the ideXlab platform.
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influence of different sugars on pullulan production and activities of α phosphoglucose Mutase udpg pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in aureobasidium pullulans y68
Carbohydrate Polymers, 2008Co-Authors: Xiaohui Duan, Lin Wang, Xianghong WangAbstract:Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.
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influence of different sugars on pullulan production and activities of α phosphoglucose Mutase udpg pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in aureobasidium pullulans y68
Carbohydrate Polymers, 2008Co-Authors: Xiaohui Duan, Zhenming Chi, Lin Wang, Xianghong WangAbstract:Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.
Arun Banerjee - One of the best experts on this subject based on the ideXlab platform.
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functional analysis of an acid adaptive dna adenine methyltransferase from helicobacter pylori 26695
PLOS ONE, 2011Co-Authors: Arun BanerjeeAbstract:HP0593 DNA-(N-6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3' and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.
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functional analysis of an acid adaptive dna adenine methyltransferase from helicobacter pylori 26695
PLOS ONE, 2011Co-Authors: Arun BanerjeeAbstract:HP0593 DNA-(N-6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3' and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.
Joanne L Turnbull - One of the best experts on this subject based on the ideXlab platform.
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use of site directed mutagenesis to identify residues specific for each reaction catalyzed by chorismate Mutase prephenate dehydrogenase from escherichia coli
Biochemistry, 1998Co-Authors: Dinesh Christendat, Vivian Saridakis, Joanne L TurnbullAbstract:Site-directed mutagenesis was performed on the bifunctional enzyme chorismate Mutase-prephenate dehydrogenase in order to identify groups important for each of the two reactions. We selected two residues for mutagenesis, Lys37 and His131, identified previously by differential peptide mapping to be essential for activity [Christendat, D., and Turnbull, J. (1996) Biochemistry 35, 4468-4479]. Kinetic studies reveal that K37Q exhibits no Mutase activity while retaining wild-type dehydrogenase activity, verifying that Lys37 plays a key role in the Mutase. By contrast His131 is not critical for the dehydrogenase; H131A is a reasonably efficient catalyst exhibiting 10% dehydrogenase and 30% Mutase activity compared to the wild-type enzyme. Chemical modification of H131A by diethyl pyrocarbonate further inactivated the dehydrogenase, suggesting that a different histidine is now accessible to modification. To identify this group, the protein's remaining eight histidines were changed to alanine or asparagine. A single substitution, H197N, decreased the dehydrogenase activity by 5 orders of magnitude while full Mutase activity was retained. In H197N, the Michaelis constants for prephenate and NAD+ and the mutant's elution profile from Sepharose-AMP were similar to those of wild-type enzyme, indicating that catalysis rather than substrate binding is altered. Log V for the dehydrogenase reaction catalyzed by H197N is pH-independent and is in contrast to wild-type enzyme, which shows a decrease in activity at low pH and pK of about 6.5. We conclude that His197 is an essential catalytic residue in the dehydrogenase reaction.
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identification of active site residues of chorismate Mutase prephenate dehydrogenase from escherichia coli
Biochemistry, 1996Co-Authors: Dinesh Christendat, Joanne L TurnbullAbstract:Chemical modification studies of the bifunctional enzyme chorismate Mutase−prephenate dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented. The reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for dehydrogenase activity and a single lysine residue essential for Mutase activity. This conclusion is based on the following evidence. (1) Hydroxylamine rapidly restores dehydrogenase activity to the DEPC-inactivated enzyme without restoring Mutase activity. (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate. (3) The reactivity of the dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the dehydrogenase reaction. (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127−135 (containing His131) and re...
Xiaohui Duan - One of the best experts on this subject based on the ideXlab platform.
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influence of different sugars on pullulan production and activities of α phosphoglucose Mutase udpg pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in aureobasidium pullulans y68
Carbohydrate Polymers, 2008Co-Authors: Xiaohui Duan, Lin Wang, Xianghong WangAbstract:Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.
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influence of different sugars on pullulan production and activities of α phosphoglucose Mutase udpg pyrophosphorylase and glucosyltransferase involved in pullulan synthesis in aureobasidium pullulans y68
Carbohydrate Polymers, 2008Co-Authors: Xiaohui Duan, Zhenming Chi, Lin Wang, Xianghong WangAbstract:Effects of different sugars on pullulan production, UDP-glucose level, and activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in Aureobasidium pullulans Y68 were examined. It was found that more pullulan was produced when the yeast strain was grown in the medium containing glucose than when it was cultivated in the medium supplementing other sugars. Our results demonstrate that when more pullulan was synthesized, less UDP-glucose was left in the cells of A. pullulans Y68. However, it was observed that more pullulan was synthesized, the cells had higher activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glycosyltransferase. Therefore, high pullulan yield is related to high activities of α-phosphoglucose Mutase, UDPG-pyrophosphorylase and glucosyltransferase in A. pullulans Y68 grown on different sugars. A pathway of pullulan biosynthesis in A. pullulan Y68 was proposed based on the results of this study and those from other researchers. This study will be helpful to metabolism-engineer the yeast strain to further enhance pullulan yield.
Bruce Ganem - One of the best experts on this subject based on the ideXlab platform.
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Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate Mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis.
Biochemistry, 1998Co-Authors: Angela Y. Lee, Sheng Zhang, Bruce Ganem, Palangpon Kongsaeree, Jon Clardy, John W. Erickson, Dong XieAbstract:Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate Mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate Mutase from Escher...
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chorismate Mutase prephenate dehydratase from escherichia coli study of catalytic and regulatory domains using genetically engineered proteins
Journal of Biological Chemistry, 1998Co-Authors: Sheng Zhang, David Wilson, Palangpon Kongsaeree, Jon Clardy, Georg Pohnert, Bruce GanemAbstract:The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate Mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full Mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked Mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The Mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the Mutase domain and the native P-protein formed dimers. Deletion of the Mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate Mutase for chorismate.
