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Christopher J. Schofield - One of the best experts on this subject based on the ideXlab platform.
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L-DELTA -(ALPHA -AMINOADIPOYL)-L-CYSTEINYL-D-VALINE SYNTHETASE : THIOESTERIFICATION OF VALINE IS NOT OBLIGATORY FOR PEPTIDE BOND FORMATION
Biochemistry, 1997Co-Authors: Chia-yang Shiau, Michael F. Byford, Jack E Baldwin, Robin T. Aplin, Christopher J. SchofieldAbstract:l-δ-(α-Aminoadipoyl)-l-cysteinyl-d-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the “thiol-template” proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides l-O-(methylserinyl)-L-Valine and l-O-(methylserinyl)-d-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97−100]. In the l-O-(methylserinyl)-L-Valine product, no significant loss of 18O was observed. However, in the l-O-(methylserinyl)-d-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bo...
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L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation.
Biochemistry, 1997Co-Authors: Chia-yang Shiau, Michael F. Byford, Jack E Baldwin, Robin T. Aplin, Christopher J. SchofieldAbstract:L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the "thiol-template" proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides L-O-(methylserinyl)-L-Valine and L-O-(methylserinyl)-D-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97-100]. In the L-O-(methylserinyl)-L-Valine product, no significant loss of 18O was observed. However, in the L-O-(methylserinyl)-D-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bond formation and the epimerization of the valine residue can both occur before formation of any thioester bond to the valine carboxylate in the biosynthesis of these dipeptides. The usual qualitative test for thioesterification of substrates to the synthetase, lability of enzyme-bound radiolabeled amino acid to performic acid, proved inconclusive in our hands. These results require a new mechanism for the enzymic synthesis of L-O-(methylserinyl)-L-Valine and L-O-(methylserinyl)-D-valine and imply that a revised mechanism for ACV synthesis is also required.
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Exchange of the valine 2-H in the biosynthesis of L-δ-(α-aminoadipoyl)-L-Cysteinyl-D-valine
Tetrahedron, 1993Co-Authors: Jack E Baldwin, Chia-yang Shiau, Michael F. Byford, Robert A. Field, Wendy J. Sobey, Christopher J. SchofieldAbstract:Abstract Incubation of [2-2H]-valine with purified ACV synthetase from both Cephalosporium acremonium and Streptomyces clavuligerus produced L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), determined by the essentially complete (>95%) loss of deuterium from the α position of the incorporated valine. Incubations with deuterium oxide/water as solvent produced ACV with significant incorporation of deuterium into the valinyl residue. These observations confirm the prior proposal that a single multifunctional enzyme is responsible for both the formation of the peptide bonds of ACV and the epimerisation of the valinyl residue.
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Studies on the exchange of valine-oxygen during the biosynthesis of δ-(L-α-Aminoadipoyl)-L-cysteinyl-D-valine.
Tetrahedron, 1992Co-Authors: Jack E Baldwin, Robert M. Adlington, Juliette W. Bird, Robert A. Field, Niamh M. O''callaghan, Christopher J. SchofieldAbstract:Abstract Incorporation of [4-2H6,18O2]-valine into δ-(L)-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), by intact cells of Cephalosporium acremonium, demonstrated the intracellular exchange of one and both valine oxygen atoms. Incubation of [18O2]-valine with the purified ACV synthetase from C. acremonium gave exclusive incorporation of a single 18O label into ACV, consistent witheffectively non-reversible formation of a covalent valinoyl-ACV synthetase intermediate under in vitro conditions.
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Exchange of valine-oxygen during the biosynthesis of δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine
J. Chem. Soc. Chem. Commun., 1991Co-Authors: Jack E Baldwin, Robert A. Field, Christopher J. SchofieldAbstract:Incubation of [18O2]valine with purified ACV synthetase from Cephalosporium acremonium gave exclusive incorporation of a single 18O label into δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), consistent with the formation of a covalent valinoyl-ACV synthetase complex.
Christophe Moinard - One of the best experts on this subject based on the ideXlab platform.
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Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?
Amino Acids, 2012Co-Authors: Servane Le Plenier, Stephane Walrand, Richard Noirt, Luc Cynober, Christophe MoinardAbstract:Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), l-leucine (1.35 g/kg, LEU, n = 10), l-citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [C-13] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P < 0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P < 0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU > CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.
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effects of leucine and citrulline versus non essential amino acids on muscle protein synthesis in fasted rat a common activation pathway
Amino Acids, 2012Co-Authors: Servane Le Plenier, Stephane Walrand, Richard Noirt, Luc Cynober, Christophe MoinardAbstract:Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), l-leucine (1.35 g/kg, LEU, n = 10), l-citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [13C] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.
Chia-yang Shiau - One of the best experts on this subject based on the ideXlab platform.
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L-DELTA -(ALPHA -AMINOADIPOYL)-L-CYSTEINYL-D-VALINE SYNTHETASE : THIOESTERIFICATION OF VALINE IS NOT OBLIGATORY FOR PEPTIDE BOND FORMATION
Biochemistry, 1997Co-Authors: Chia-yang Shiau, Michael F. Byford, Jack E Baldwin, Robin T. Aplin, Christopher J. SchofieldAbstract:l-δ-(α-Aminoadipoyl)-l-cysteinyl-d-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the “thiol-template” proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides l-O-(methylserinyl)-L-Valine and l-O-(methylserinyl)-d-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97−100]. In the l-O-(methylserinyl)-L-Valine product, no significant loss of 18O was observed. However, in the l-O-(methylserinyl)-d-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bo...
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L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine synthetase: thioesterification of valine is not obligatory for peptide bond formation.
Biochemistry, 1997Co-Authors: Chia-yang Shiau, Michael F. Byford, Jack E Baldwin, Robin T. Aplin, Christopher J. SchofieldAbstract:L-delta-(alpha-Aminoadipoyl)-L-cysteinyl-D-valine (ACV) synthetase is probably the simplest known peptide synthetase in terms of the number of reactions catalyzed. In the "thiol-template" proposal for nonribosomal peptide synthesis, a key step is transfer of aminoacyl groups derived from the substrates to enzyme-bound thiols prior to peptide bond formation. No incorporation of 18O was seen in AMP isolated from the reaction mixture when di[18O]valine was incubated with relatively large amounts of active synthetase and MgATP. We therefore utilized di[18O]valine as a substrate for the biosynthesis of the diastereomeric dipeptides L-O-(methylserinyl)-L-Valine and L-O-(methylserinyl)-D-valine [Shiau, C.-Y., Baldwin, J. E., Byford, M. F., Sobey, W. J., & Schofield, C. J. (1995) FEBS Lett. 358, 97-100]. In the L-O-(methylserinyl)-L-Valine product, no significant loss of 18O was observed. However, in the L-O-(methylserinyl)-D-valine product, a significant loss of one or both 18O labels was observed. Thus, both peptide bond formation and the epimerization of the valine residue can both occur before formation of any thioester bond to the valine carboxylate in the biosynthesis of these dipeptides. The usual qualitative test for thioesterification of substrates to the synthetase, lability of enzyme-bound radiolabeled amino acid to performic acid, proved inconclusive in our hands. These results require a new mechanism for the enzymic synthesis of L-O-(methylserinyl)-L-Valine and L-O-(methylserinyl)-D-valine and imply that a revised mechanism for ACV synthesis is also required.
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Exchange of the valine 2-H in the biosynthesis of L-δ-(α-aminoadipoyl)-L-Cysteinyl-D-valine
Tetrahedron, 1993Co-Authors: Jack E Baldwin, Chia-yang Shiau, Michael F. Byford, Robert A. Field, Wendy J. Sobey, Christopher J. SchofieldAbstract:Abstract Incubation of [2-2H]-valine with purified ACV synthetase from both Cephalosporium acremonium and Streptomyces clavuligerus produced L-δ-(α-aminoadipoyl)-L-cysteinyl-D-valine (ACV), determined by the essentially complete (>95%) loss of deuterium from the α position of the incorporated valine. Incubations with deuterium oxide/water as solvent produced ACV with significant incorporation of deuterium into the valinyl residue. These observations confirm the prior proposal that a single multifunctional enzyme is responsible for both the formation of the peptide bonds of ACV and the epimerisation of the valinyl residue.
Servane Le Plenier - One of the best experts on this subject based on the ideXlab platform.
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Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?
Amino Acids, 2012Co-Authors: Servane Le Plenier, Stephane Walrand, Richard Noirt, Luc Cynober, Christophe MoinardAbstract:Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), l-leucine (1.35 g/kg, LEU, n = 10), l-citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [C-13] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P < 0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P < 0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU > CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.
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effects of leucine and citrulline versus non essential amino acids on muscle protein synthesis in fasted rat a common activation pathway
Amino Acids, 2012Co-Authors: Servane Le Plenier, Stephane Walrand, Richard Noirt, Luc Cynober, Christophe MoinardAbstract:Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), l-leucine (1.35 g/kg, LEU, n = 10), l-citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [13C] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.
Jin Hwan Park - One of the best experts on this subject based on the ideXlab platform.
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fed batch culture of escherichia coli for l valine production based on in silico flux response analysis
Biotechnology and Bioengineering, 2011Co-Authors: Jin Hwan Park, Tae Yong Kim, Kwang Ho Lee, Sang Yup LeeAbstract:We have previously reported the development of a 100% genetically defined engineered Escherichia coli strain capable of producing L-Valine from glucose with a high yield of 0.38 g L-Valine per gram glucose (0.58 mol L-Valine per mol glucose) by batch culture. Here we report a systems biological strategy of employing flux response analysis in bioprocess development using L-Valine production by fed-batch culture as an example. Through the systems-level analysis, the source of ATP was found to be important for efficient L-Valine production. There existed a trade-off between L-Valine production and biomass formation, which was optimized for the most efficient L-Valine production. Furthermore, acetic acid feeding strategy was optimized based on flux response analysis. The final fed-batch cultivation strategy allowed production of 32.3 g/L L-Valine, the highest concentration reported for E. coli. This approach of employing systems-level analysis of metabolic fluxes in developing fed-batch cultivation strategy would also be applicable in developing strategies for the efficient production of other bioproducts. Biotechnol. Bioeng. 2011; 108:934–946. © 2010 Wiley Periodicals, Inc.
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Fed‐batch culture of Escherichia coli for L‐valine production based on in silico flux response analysis
Biotechnology and bioengineering, 2010Co-Authors: Jin Hwan Park, Tae Yong Kim, Kwang Ho Lee, Sang Yup LeeAbstract:We have previously reported the development of a 100% genetically defined engineered Escherichia coli strain capable of producing L-Valine from glucose with a high yield of 0.38 g L-Valine per gram glucose (0.58 mol L-Valine per mol glucose) by batch culture. Here we report a systems biological strategy of employing flux response analysis in bioprocess development using L-Valine production by fed-batch culture as an example. Through the systems-level analysis, the source of ATP was found to be important for efficient L-Valine production. There existed a trade-off between L-Valine production and biomass formation, which was optimized for the most efficient L-Valine production. Furthermore, acetic acid feeding strategy was optimized based on flux response analysis. The final fed-batch cultivation strategy allowed production of 32.3 g/L L-Valine, the highest concentration reported for E. coli. This approach of employing systems-level analysis of metabolic fluxes in developing fed-batch cultivation strategy would also be applicable in developing strategies for the efficient production of other bioproducts. Biotechnol. Bioeng. 2011; 108:934–946. © 2010 Wiley Periodicals, Inc.
