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Gerhard H. Braus - One of the best experts on this subject based on the ideXlab platform.
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A Glutamate Residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Norbert Sträter, Gerhard H. BrausAbstract:Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid Residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid Residues. The resulting enzymes exhibit the necessity of these Residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
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A Glutamate Residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions (Claisen rearrangementysite-directed mutagenesis)
1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract:Chorismate mutase acts at the first branch- point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutaseyprephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid Residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr- 242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid Residues. The resulting enzymes exhibit the neces- sity of these Residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest ac- tivity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
Gebre Woldegiorgis - One of the best experts on this subject based on the ideXlab platform.
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a single amino acid change substitution of the conserved glu 590 with alanine in the c terminal domain of rat liver carnitine palmitoyltransferase i increases its malonyl coa sensitivity close to that observed with the muscle isoform of the enzyme
Journal of Biological Chemistry, 2003Co-Authors: Laura Napal, Michelle Treber, Jia Dai, Diego Haro, Pedro F Marrero, Gebre WoldegiorgisAbstract:Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long-chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the highly conserved C-terminal Glutamate Residue, Glu-590, on catalysis and malonyl-CoA sensitivity, we separately changed the Residue to alanine, lysine, glutamine, and aspartate. Substitution of Glu-590 with aspartate, a negatively charged amino acid with only one methyl group less than the Glutamate Residue in the wild-type enzyme, resulted in complete loss in the activity of the liver isoform of CPTI (L-CPTI). A change of Glu-590 to alanine, glutamine, and lysine caused a significant 9- to 16-fold increase in malonyl-CoA sensitivity but only a partial decrease in catalytic activity. Substitution of Glu-590 with neutral uncharged Residues (alanine and glutamine) and/or a basic positively charged Residue (lysine) significantly increased L-CPTI malonyl-CoA sensitivity to the level observed with the muscle isoform of the enzyme, suggesting the importance of neutral and/or positive charges in the switch of the kinetic properties of L-CPTI to the muscle isoform of CPTI. Since a conservative substitution of Glu-590 to aspartate but not glutamine resulted in complete loss in activity, we suggest that the longer side chain of Glutamate is essential for catalysis and malonyl-CoA sensitivity. This is the first demonstration whereby a single Residue mutation in the C-terminal region of the liver isoform of CPTI resulted in a change of its kinetic properties close to that observed with the muscle isoform of the enzyme and provides the rationale for the high malonyl-CoA sensitivity of muscle CPTI compared with the liver isoform of the enzyme.
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identification by mutagenesis of conserved arginine and Glutamate Residues in the c terminal domain of rat liver carnitine palmitoyltransferase i that are important for catalytic activity and malonyl coa sensitivity
Journal of Biological Chemistry, 2003Co-Authors: Michelle Treber, Gebre WoldegiorgisAbstract:Abstract Carnitine palmitoyltransferase I (CPTI) catalyzes the conversion of long chain fatty acyl-CoAs to acylcarnitines in the presence of l-carnitine. To determine the role of the conserved Glutamate Residue, Glu-603, on catalysis and malonyl-CoA sensitivity, we separately changed the Residue to alanine, histidine, glutamine, and aspartate. Substitution of Glu-603 with alanine or histidine resulted in complete loss of L-CPTI activity. A change of Glu-603 to glutamine caused a significant decrease in catalytic activity and malonyl-CoA sensitivity. Substitution of Glu-603 with aspartate, a negatively charged amino acid with only one methyl group less than the Glutamate Residue in the wild type enzyme, resulted in partial loss in CPTI activity and a 15-fold decrease in malonyl-CoA sensitivity. The mutant L-CPTI with a replacement of the conserved Arg-601 or Arg-606 with alanine also showed over 40-fold decrease in malonyl-CoA sensitivity, suggesting that these two conserved Residues may be important for substrate and inhibitor binding. Since a conservative substitution of Glu-603 to aspartate or glutamine resulted in partial loss of activity and malonyl-CoA sensitivity, it further suggests that the negative charge and the longer side chain of Glutamate are essential for catalysis and malonyl-CoA sensitivity. We predict that this region of L-CPTI spanning these conserved C-terminal Residues may be the region of the protein involved in binding the CoA moiety of palmitoyl-CoA and malonyl-CoA and/or the putative low affinity acyl-CoA/malonyl-CoA binding site.
Liqun Jiang - One of the best experts on this subject based on the ideXlab platform.
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filamentous cyanobacteria triples oil production in seawater based medium supplemented with industrial waste monosodium Glutamate Residue
Biotechnology for Biofuels, 2019Co-Authors: Liqun Jiang, Jiongming Sun, Changliang Nie, Jackson Jenkins, Haiyan PeiAbstract:To overcome the daunting technical and economic barriers of algal biofuels, we evaluated whether seawater can be a viable medium for economically producing filamentous Spirulina subsalsa as feedstock, using monosodium Glutamate Residue (MSGR) produced by the Glutamate extraction process as an inexpensive nutrient source. Spirulina subsalsa cannot grow in pure seawater, but exhibited faster biomass accumulation in seawater supplemented with MSGR than in freshwater medium (modified Zarrouk medium). Introducing seawater into media ensured this cyanobacterium obtained high lipid productivity (120 mg/L/day) and suffered limited bacterial infections during growth. Moreover, the yields of protein, carotenoids and phytols were also improved in seawater mixed with MSGR. S. subsalsa exhibited high biomass and lipid productivity in bag bioreactors with 5- and 10-L medium, demonstrating the potential of this cultivation method for scaling up. Moreover, seawater can produce more biomass through medium reuse. Reused seawater medium yielded 72% of lipid content compared to pristine medium. The reason that S. subsalsa grew well in seawater with MSGR is its proficient adaptation to salinity, which included elongation and desaturation of fatty acids, accumulation of lysine and methionine, and secretion of sodium. The nutrients provided by MSGR, like organic materials, played an important role in these responses. Spirulina subsalsa has an efficient system to adapt to saline ambiance in seawater. When supplemented with MSGR, seawater is a great potential medium to produce S. subsalsa in large scale as biofuel feedstock. Meanwhile, value-added products can be derived from the ample protein and pigments that can broaden the range of biomass application and improve this biorefinery economics.
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Filamentous cyanobacteria triples oil production in seawater-based medium supplemented with industrial waste: monosodium Glutamate Residue
BMC, 2019Co-Authors: Liqun Jiang, Jiongming Sun, Changliang Nie, Jackson Jenkins, Haiyan PeiAbstract:Abstract Background To overcome the daunting technical and economic barriers of algal biofuels, we evaluated whether seawater can be a viable medium for economically producing filamentous Spirulina subsalsa as feedstock, using monosodium Glutamate Residue (MSGR) produced by the Glutamate extraction process as an inexpensive nutrient source. Results Spirulina subsalsa cannot grow in pure seawater, but exhibited faster biomass accumulation in seawater supplemented with MSGR than in freshwater medium (modified Zarrouk medium). Introducing seawater into media ensured this cyanobacterium obtained high lipid productivity (120 mg/L/day) and suffered limited bacterial infections during growth. Moreover, the yields of protein, carotenoids and phytols were also improved in seawater mixed with MSGR. S. subsalsa exhibited high biomass and lipid productivity in bag bioreactors with 5- and 10-L medium, demonstrating the potential of this cultivation method for scaling up. Moreover, seawater can produce more biomass through medium reuse. Reused seawater medium yielded 72% of lipid content compared to pristine medium. The reason that S. subsalsa grew well in seawater with MSGR is its proficient adaptation to salinity, which included elongation and desaturation of fatty acids, accumulation of lysine and methionine, and secretion of sodium. The nutrients provided by MSGR, like organic materials, played an important role in these responses. Conclusion Spirulina subsalsa has an efficient system to adapt to saline ambiance in seawater. When supplemented with MSGR, seawater is a great potential medium to produce S. subsalsa in large scale as biofuel feedstock. Meanwhile, value-added products can be derived from the ample protein and pigments that can broaden the range of biomass application and improve this biorefinery economics
Georg Schnappauf - One of the best experts on this subject based on the ideXlab platform.
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A Glutamate Residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Norbert Sträter, Gerhard H. BrausAbstract:Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid Residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid Residues. The resulting enzymes exhibit the necessity of these Residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
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A Glutamate Residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions (Claisen rearrangementysite-directed mutagenesis)
1997Co-Authors: Georg Schnappauf, William N. Lipscomb, Gerhard H. BrausAbstract:Chorismate mutase acts at the first branch- point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutaseyprephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid Residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr- 242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid Residues. The resulting enzymes exhibit the neces- sity of these Residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest ac- tivity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.
Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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Oxygen Exchange between Acetate and the Catalytic Glutamate Residue in Glutaconate CoA-transferase from Acidaminococcus fermentans IMPLICATIONS FOR THE MECHANISM OF CoA-ESTER HYDROLYSIS
Journal of Biological Chemistry, 1999Co-Authors: Thorsten Selmer, Wolfgang BuckelAbstract:Abstract The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic Glutamate Residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [18O2]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [18O2]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic Glutamate Residue by aspartate, no 18O uptake from H2 18O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.
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identification of Glutamate β54 as the covalent catalytic Residue in the active site of glutaconate coa transferase from acidaminococcus fermentans
FEBS Letters, 1995Co-Authors: Matthias Mack, Wolfgang BuckelAbstract:In the course of Glutamate fermentation by Acidaminococcus fermentans glutaconate coenzyme A-transferase catalyzes the transfer of CoAS− from acetyl-CoA to (R)-2-hydroxyg;utarate, forming (R)-2-hydroxyglutaryl-CoA. Glutamate (E) 54 of the β-subunit was postulated to be directly involved in catalysis by formation of a CoASH ester intermediate [(1994) Eur. J. Biochem., in press]. In order to prove this preliminary result, the following mutations, βE54A, βE64A, βE54Q and βE54D, were introduced by mismatch oligonucleotide priming. As expected, βE54A was inactive (0.02% of the wild-type), whereas βE64A and βE54D were active, 30% and >7%, respectively. However, no CoASH intermediate was detected in the latter mutant, indicating a change in the catalytic mechanism. The activity of the βE54Q mutant increased from 1% to almost 100% upon incubation with acetyl-CoA and glutaconate at 37°C within 40 h. Hence, the substrates induced the conversion of the mutant glutamine Residue into the Glutamate Residue of the wild-type enzyme.