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Martin R. Webb - One of the best experts on this subject based on the ideXlab platform.
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mechanism of inorganic Phosphate interaction with Phosphate Binding Protein from escherichia coli
Biochemistry, 1998Co-Authors: Martin Brune, John E. T. Corrie, Jackie L Hunter, Steven Howell, Stephen R Martin, Theodore L Hazlett, Martin R. WebbAbstract:The mechanism of Pi interaction with Phosphate Binding Protein of Escherichia coli has been investigated using the A197C mutant Protein labeled with a coumarin fluorophore (MDCC−PBP), which gives a fluorescence change on Binding Pi. A pure preparation of MDCC−PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi Binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC−PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi Binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after Binding, concomitant with a conformational change of the Protein that closes the cleft containing the Pi bind...
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crystal structure of Phosphate Binding Protein labeled with a coumarin fluorophore a probe for inorganic Phosphate
Biochemistry, 1998Co-Authors: Miriam Hirshberg, Lesley Lloyd Haire, Nishi Vasisht, Martin Brune, John E. T. Corrie, Kim Henrick, Martin R. WebbAbstract:Crystal structures are presented for the A197C mutant of Escherichia coli Phosphate Binding Protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both Proteins are complexed with inorganic Phosphate. The latter molecule, MDCC−PBP, exhibits a large increase in fluorescence on Binding inorganic Phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic Phosphate Binding are interpreted in terms of the specific interactions of MDCC with the Protein. The structure helps to explain why this particular label gives a high-fluorescence state on Binding inorganic Phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on Proteins.
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mechanism of inorganic Phosphate interaction with Phosphate Binding Protein from escherichia coli
Biochemistry, 1998Co-Authors: Martin Brune, John E. T. Corrie, Jackie L Hunter, Steven Howell, Stephen R Martin, Theodore L Hazlett, Martin R. WebbAbstract:The mechanism of Pi interaction with Phosphate Binding Protein of Escherichia coli has been investigated using the A197C mutant Protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on Binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi Binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi Binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after Binding, concomitant with a conformational change of the Protein that closes the cleft containing the Pi Binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi Binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on Binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the Protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the Protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.
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kinetics of nucleoside triPhosphate cleavage and Phosphate release steps by associated rabbit skeletal actomyosin measured using a novel fluorescent probe for Phosphate
Biochemistry, 1997Co-Authors: Howard D White, Betty Belknap, Martin R. WebbAbstract:We have measured the kinetics of inorganic Phosphate (Pi) release during a single turnover of actomyosin nucleoside triPhosphate (NTP) hydrolysis using a double-mixing stopped-flow spectrofluorometer, at very low ionic strength to increase the affinity of myosin-ATP and myosin-ADP-Pi to actin. Myosin subfragment 1 and a series of nucleoside triPhosphates were mixed and incubated for approximately 1-10 s to allow NTP to bind to myosin and generate a steady state mixture of myosin-NTP and myosin-NDP-Pi. The steady state intermediates were then mixed with actin. The kinetics of Pi release were measured using a fluorescent probe for Pi, based on a Phosphate Binding Protein [Brune et al. (1994) Biochemistry 33, 8262-8271]. These data are correlated with quenched-flow data, where the extent of the rapid burst of hydrolysis during the first turnover of ATP hydrolysis was followed by chemical quenching of the reaction mix at various times after rapidly mixing ATP and myosin subfragment 1. From the double-mixing actomyosin measurements, the kinetics of Pi release are biphasic. The fast phase corresponds to Pi release from the associated actomyosin-ADP-Pi complex. The slow phase measures the rate of the cleavage step on associated actomyosin. At saturating actin, there is a correlation between the amplitude of the fast phase and the size of the Pi burst observed by quenched flow in the absence of actin: the size of this phase corresponds to the amount of myosin-ADP-Pi formed during the first mix. For ATP at 20 degrees C the rate of the Pi release step is 75 (+/-5) s-1, 25-fold larger than the cleavage step, which is the rate-limiting step of actomyosin ATP hydrolysis at saturating actin. The rate constant of Pi release varies only slightly with nucleoside structure. The rate constant of the slow phase of the Pi release (measuring cleavage) is highly dependent upon the structure of the NTP substrate.
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atpase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres a real time Phosphate assay
The Journal of Physiology, 1997Co-Authors: Rod K Chillingworth, Martin Brune, John E. T. Corrie, Martin R. Webb, David R Trentham, Michael A. FerencziAbstract:1. The rate of appearance of inorganic Phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the Phosphate-Binding Protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.
Tohru Yoshimura - One of the best experts on this subject based on the ideXlab platform.
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conserved pyridoxal 5 Phosphate Binding Protein yggs impacts amino acid metabolism through pyridoxine 5 Phosphate in escherichia coli
Applied and Environmental Microbiology, 2019Co-Authors: Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Kana Yamamoto, Ran Hori, Diana M Downs, Tohru YoshimuraAbstract:ABSTRACT Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5′-Phosphate (PLP)-Binding Protein (PLPBP) family. Recent studies suggested a role for this Protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this Protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5′-Phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli. The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coli. IMPORTANCE Recent studies showed that deletion or mutation of members of the YggS Protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS Protein family.
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ophthalmic acid accumulation in an escherichia coli mutant lacking the conserved pyridoxal 5 Phosphate Binding Protein yggs
Journal of Bioscience and Bioengineering, 2016Co-Authors: Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Tohru YoshimuraAbstract:Escherichia coli YggS is a highly conserved pyridoxal 5'-Phosphate (PLP)-Binding Protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.
Martin Brune - One of the best experts on this subject based on the ideXlab platform.
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mechanism of inorganic Phosphate interaction with Phosphate Binding Protein from escherichia coli
Biochemistry, 1998Co-Authors: Martin Brune, John E. T. Corrie, Jackie L Hunter, Steven Howell, Stephen R Martin, Theodore L Hazlett, Martin R. WebbAbstract:The mechanism of Pi interaction with Phosphate Binding Protein of Escherichia coli has been investigated using the A197C mutant Protein labeled with a coumarin fluorophore (MDCC−PBP), which gives a fluorescence change on Binding Pi. A pure preparation of MDCC−PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi Binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC−PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi Binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after Binding, concomitant with a conformational change of the Protein that closes the cleft containing the Pi bind...
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crystal structure of Phosphate Binding Protein labeled with a coumarin fluorophore a probe for inorganic Phosphate
Biochemistry, 1998Co-Authors: Miriam Hirshberg, Lesley Lloyd Haire, Nishi Vasisht, Martin Brune, John E. T. Corrie, Kim Henrick, Martin R. WebbAbstract:Crystal structures are presented for the A197C mutant of Escherichia coli Phosphate Binding Protein (PBP) and the same mutant labeled at Cys197 with N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Both Proteins are complexed with inorganic Phosphate. The latter molecule, MDCC−PBP, exhibits a large increase in fluorescence on Binding inorganic Phosphate. The resulting high-fluorescence state of the coumarin and the ability of this coumarin to monitor the conformational changes associated with inorganic Phosphate Binding are interpreted in terms of the specific interactions of MDCC with the Protein. The structure helps to explain why this particular label gives a high-fluorescence state on Binding inorganic Phosphate, while several other related labels do not, and hence aids our general understanding of environmentally sensitive fluorescence probes on Proteins.
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mechanism of inorganic Phosphate interaction with Phosphate Binding Protein from escherichia coli
Biochemistry, 1998Co-Authors: Martin Brune, John E. T. Corrie, Jackie L Hunter, Steven Howell, Stephen R Martin, Theodore L Hazlett, Martin R. WebbAbstract:The mechanism of Pi interaction with Phosphate Binding Protein of Escherichia coli has been investigated using the A197C mutant Protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on Binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi Binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi Binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after Binding, concomitant with a conformational change of the Protein that closes the cleft containing the Pi Binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi Binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on Binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the Protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the Protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.
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atpase kinetics on activation of rabbit and frog permeabilized isometric muscle fibres a real time Phosphate assay
The Journal of Physiology, 1997Co-Authors: Rod K Chillingworth, Martin Brune, John E. T. Corrie, Martin R. Webb, David R Trentham, Michael A. FerencziAbstract:1. The rate of appearance of inorganic Phosphate (Pi) and hence the ATPase activity of rabbit psoas muscle in single permeabilized muscle fibres initially in rigor was measured following laser flash photolysis of the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP) in the presence and absence of Ca2+. Pi appearance was monitored from the fluorescence signal of a Pi-sensitive probe, MDCC-PBP, a coumarin-labelled A197C mutant of the Phosphate-Binding Protein from Escherichia coli. Fibres were immersed in oil to optimize the fluorescence signal and to obviate diffusion problems. The ATPase activity was also measured under similar conditions from the rate of NADH disappearance using an NADH-linked coupled enzyme assay. 2. On photolysis of NPE-caged ATP in the presence of Ca2+ at 20 degrees C, the fluorescence increase of MDCC-PBP was non-linear with time. ATPase activity was 41 s-1 in the first turnover based on a myosin subfragment 1 concentration of 150 microM. This was calculated from a linear regression of the fluorescence signal reporting 20-150 microM of Pi release. Tension was at 67% of its isometric level by the time 150 microM Pi was released. ATPase activities were 36 and 31 s-1 for Pi released in the ranges of 150-300 microM and 300-450 microM, respectively. The ATPase activity had a Q10 value of 2.9 based on measurements at 5, 12 and 20 degrees C. 3. An NADH-linked assay showed the ATPase activity had a lower limit of 12.7 s-1 at 20 degrees C. The response to photolytic release of ADP showed that the rate of NADH disappearance was partially limited by the flux through the coupled reactions. Simulations indicated that the linked assay data were consistent with an initial ATPase activity of 40 s-1. 4. On photolysis of NPE-caged ATP in the absence of Ca2+ the ATPase activity was 0.11 s-1 at 20 degrees C with no discernible rapid transient phase of Pi release during the first turnover of the ATPase. 5. To avoid the rigor state, the ATPase rate in the presence of Ca2+ was also measured on activation from the relaxed state by photolytic release of Ca2+ from a caged Ca2+ compound, nitrophenyl-EGTA. At 5 degrees C the ATPase rate was 5.8 and 4.0 s-1 in the first and second turnovers, respectively. These rates are comparable to those when NPE-caged ATP was used. 6. The influence of ADP and Pi on the ATPase activities was measured using the MDCC-PBP and NADH-linked assays, respectively. ADP (0.5 mM) decreased the initial ATPase rate by 23%. Pi (10 mM) had no significant effect. Inhibition by ADP, formed during ATP hydrolysis, contributed to the decrease of ATPase activity with time. 7. The MDCC-PBP assay and NPE-caged ATP were used to measure the ATPase rate in single permeabilized muscle fibres of the semitendinosus muscle of the frog. At 5 degrees C in the presence of Ca2+ the ATPase activity was biphasic being 15.0 s-1 during the first turnover (based on 180 microM myosin subfragment 1). Tension was 74% of its isometric level by the time 180 microM Pi was released. During the third turnover the ATPase rate decreased to about 20% of that during the first turnover. 8. ATPase activity in isometric rabbit muscle fibres during the first few turnovers is about an order of magnitude greater than that when a steady state is reached. Possible reasons and the consequences for understanding the mechanism of muscular contraction are discussed.
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direct real time measurement of rapid inorganic Phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 atpase
Biochemistry, 1994Co-Authors: Martin Brune, John E. T. Corrie, Jackie L Hunter, Martin R. WebbAbstract:: A probe has been developed that can rapidly measure micromolar concentrations of inorganic Phosphate (Pi), in particular to follow the release of Pi in real time from enzymes such as phosphatases. Its application is described to investigate the mechanism of actomyosin subfragment 1 ATPase. The probe uses the A197C mutant of Escherichia coli Phosphate Binding Protein (PBP), generated by oligonucleotide-directed mutagenesis. A new fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), was attached to the single cysteine to produce the reporter molecule that was purified free of unlabeled Protein and unattached MDCC. The labeled Protein has an excitation maximum at 425 nm and emission maximum at 474 nm in the absence of Pi, shifting to 464 nm with a 5.2-fold increase in fluorescence (lambda max/lambda max) when complexed with Pi at pH 7.0, low ionic strength, 22 degrees C. The fluorescence increase is not much altered by change to pH 8 or by increase in ionic strength to 1 M. Pi binds tightly (Kd approximately 0.1 microM) and rapidly (1.36 x 10(8) M-1 s-1) and the dissociation rate constant is 21 s-1, at pH 7.0, low ionic strength, 22 degrees C. A variety of Phosphate esters were tested to investigate the specificity of the MDCC-PBP and none gave a significant fluorescence increase at 100 microM or higher concentration. ATP weakly inhibited the Pi-induced fluorescence change, indicating that it binds at least 3000-fold weaker than Pi. Because Pi is a widespread contaminant, the probe is used in conjunction with a "Pi mop", consisting of 7-methylguanosine and purine nucleoside phosphorylase, to remove free Pi from solutions by its conversion to ribose 1-Phosphate. Because the equilibrium constant of this reaction is > 100, free Pi can be reduced below 0.1 microM. The probe was used to measure the rate of Pi release during a single turnover of ATP hydrolysis with actomyosin subfragment 1 from rabbit skeletal muscle, to determine to what extent Pi release contributes to the rate limitation of this ATPase. Using a stopped-flow apparatus, a small lag prior to rapid Pi release was detected at pH 7.0, low ionic strength, between 5 and 22 degrees C at both high and low [ATP]. For measurements of a single turnover at low [ATP], the observed rate increased with [actin], showing saturation with a Km with respect to actin of 26 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
Kyu Y Rhee - One of the best experts on this subject based on the ideXlab platform.
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aspartate aminotransferase rv3722c governs aspartate dependent nitrogen metabolism in mycobacterium tuberculosis
Nature Communications, 2020Co-Authors: Robert S Jansen, Lungelo Mandyoli, Ryan N Hughes, Shoko Wakabayashi, Jessica T Pinkham, Bruna Selbach, Kristine M Guinn, Eric J Rubin, James C Sacchettini, Kyu Y RheeAbstract:Gene rv3722c of Mycobacterium tuberculosis is essential for in vitro growth, and encodes a putative pyridoxal Phosphate-Binding Protein of unknown function. Here we use metabolomic, genetic and structural approaches to show that Rv3722c is the primary aspartate aminotransferase of M. tuberculosis, and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Rv3722c deficiency leads to virulence attenuation in macrophages and mice. Our results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in M. tuberculosis.
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rv3722c governs aspartate dependent nitrogen metabolism in mycobacterium tuberculosis
bioRxiv, 2019Co-Authors: Robert S Jansen, Lungelo Mandyoli, Ryan N Hughes, Shoko Wakabayashi, Jessica T Pinkham, Bruna Selbach, Kristine M Guinn, Eric J Rubin, James C Sacchettini, Kyu Y RheeAbstract:Abstract Organisms are defined by their genomes, yet many distinguishing features of a given organism are encoded by genes that are functionally unannotated. Mycobacterium tuberculosis (Mtb), the leading cause of death due to a single microbe, co-evolved with humans as its only known natural reservoir, yet the factors mediating Mtb’s pathogenicity remain incompletely defined. rv3722c is a gene of unknown function predicted to encode a pyridoxal Phosphate Binding Protein and to be essential for in vitro growth of Mtb. Using metabolomic, genetic and structural approaches, we show that Rv3722c is the primary aspartate aminotransferase of Mtb and mediates an essential but underrecognized role in metabolism: nitrogen distribution. Together with the attenuation of Rv3722c-deficient Mtb in macrophages and mice, these results identify aspartate biosynthesis and nitrogen distribution as potential species-selective drug targets in Mtb.
Tomokazu Ito - One of the best experts on this subject based on the ideXlab platform.
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conserved pyridoxal 5 Phosphate Binding Protein yggs impacts amino acid metabolism through pyridoxine 5 Phosphate in escherichia coli
Applied and Environmental Microbiology, 2019Co-Authors: Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Kana Yamamoto, Ran Hori, Diana M Downs, Tohru YoshimuraAbstract:ABSTRACT Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5′-Phosphate (PLP)-Binding Protein (PLPBP) family. Recent studies suggested a role for this Protein family in the homeostasis of vitamin B6 and amino acids. The deletion or mutation of a member of this Protein family causes pleiotropic effects in many organisms and is causative of vitamin B6-dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5′-Phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli. The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coli. IMPORTANCE Recent studies showed that deletion or mutation of members of the YggS Protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS Protein family.
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ophthalmic acid accumulation in an escherichia coli mutant lacking the conserved pyridoxal 5 Phosphate Binding Protein yggs
Journal of Bioscience and Bioengineering, 2016Co-Authors: Tomokazu Ito, Ayako Yamauchi, Hisashi Hemmi, Tohru YoshimuraAbstract:Escherichia coli YggS is a highly conserved pyridoxal 5'-Phosphate (PLP)-Binding Protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.