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Jia Jeong - One of the best experts on this subject based on the ideXlab platform.
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roles of Alanine Dehydrogenase and induction of its gene in mycobacterium smegmatis under respiration inhibitory conditions
Journal of Bacteriology, 2018Co-Authors: Jia Jeong, Sae Woong Park, Dahae Yoon, Suhkmann Kim, Ho Young KangAbstract:Here we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3 cytochrome c oxidase) or treatment with KCN resulted in the induction of ald encoding Alanine Dehydrogenase in Mycobacterium smegmatis A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+ pool toward a more reduced state, which in turn leads to an increase in cellular levels of Alanine by Ald catalyzing the conversion of pyruvate to Alanine with the concomitant oxidation of NADH to NAD+ The induction of ald expression under respiration-inhibitory conditions in M. smegmatis is mediated by the Alanine-responsive AldR transcriptional regulator. The growth defect of M. smegmatis by respiration inhibition was exacerbated by inactivation of the ald gene, suggesting that Ald is beneficial to M. smegmatis in its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+ homeostasis. The low susceptibility of M. smegmatis to bcc1 complex inhibitors appears to be, at least in part, attributable to the high expression level of the bd quinol oxidase in M. smegmatis when the bcc1-aa3 branch of the ETC is inactivated.IMPORTANCE We demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of the ald gene encoding Alanine Dehydrogenase in Mycobacterium smegmatis Furthermore, the importance of Ald in NADH/NAD+ homeostasis during the adaptation of M. smegmatis to severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.
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regulation mechanism of the ald gene encoding Alanine Dehydrogenase in mycobacterium smegmatis and mycobacterium tuberculosis by the lrp asnc family regulator aldr
Journal of Bacteriology, 2015Co-Authors: Jia Jeong, Jaekyung HyunAbstract:In the presence of Alanine, AldR, which belongs to the Lrp/AsnC family of transcriptional regulators and regulates ald encoding Alanine Dehydrogenase in Mycobacterium smegmatis, changes its quaternary structure from a homodimer to an octamer with an open-ring conformation. Four AldR-binding sites (O2, O1, O4, and O3) with a consensus sequence of GA/T-N2-NWW/WWN-N2-A/TC were identified upstream of the M. smegmatis ald gene by means of DNase I footprinting analysis. O2, O1, and O4 are required for the induction of ald expression by Alanine, while O3 is directly involved in the repression of ald expression. In addition to O3, both O1 and O4 are also necessary for full repression of ald expression in the absence of Alanine, due to cooperative binding of AldR dimers to O1, O4, and O3. Binding of a molecule of the AldR octamer to the ald control region was demonstrated to require two AldR-binding sites separated by three helical turns between their centers and one additional binding site that is in phase with the two AldR-binding sites. The cooperative binding of AldR dimers to DNA requires three AldR-binding sites that are aligned with a periodicity of three helical turns. The aldR gene is negatively autoregulated independently of Alanine. Comparative analysis of ald expression of M. smegmatis and Mycobacterium tuberculosis in conjunction with sequence analysis of both ald control regions led us to suggest that the expression of the ald genes in both mycobacterial species is regulated by the same mechanism. IMPORTANCE In mycobacteria, Alanine Dehydrogenase (Ald) is the enzyme required both to utilize Alanine as a nitrogen source and to grow under hypoxic conditions by maintaining the redox state of the NADH/NAD+ pool. Expression of the ald gene was reported to be regulated by the AldR regulator that belongs to the Lrp/AsnC (feast/famine) family, but the underlying mechanism was unknown. This study revealed the regulation mechanism of ald in Mycobacterium smegmatis and Mycobacterium tuberculosis. Furthermore, a generalized arrangement pattern of cis-acting regulatory sites for Lrp/AsnC (feast/famine) family regulators is suggested in this study.
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regulation of the ald gene encoding Alanine Dehydrogenase by aldr in mycobacterium smegmatis
Journal of Bacteriology, 2013Co-Authors: Jia Jeong, Eunyoung Baek, Si Wouk Kim, Jongsoon ChoiAbstract:The regulatory gene aldR was identified 95 bp upstream of the ald gene encoding l-Alanine Dehydrogenase in Mycobacterium smegmatis. The AldR protein shows sequence similarity to the regulatory proteins of the Lrp/AsnC family. Using an aldR deletion mutant, we demonstrated that AldR serves as both activator and repressor for the regulation of ald gene expression, depending on the presence or absence of l-Alanine. The purified AldR protein exists as a homodimer in the absence of l-Alanine, while it adopts the quaternary structure of a homohexamer in the presence of l-Alanine. The binding affinity of AldR for the ald control region was shown to be increased significantly by l-Alanine. Two AldR binding sites (O1 and O2) with the consensus sequence GA-N2-ATC-N2-TC and one putative AldR binding site with the sequence GA-N2-GTT-N2-TC were identified upstream of the ald gene. Alanine and cysteine were demonstrated to be the effector molecules directly involved in the induction of ald expression. The cellular level of l-Alanine was shown to be increased in M. smegmatis cells grown under hypoxic conditions, and the hypoxic induction of ald expression appears to be mediated by AldR, which senses the intracellular level of Alanine.
David W. Emerich - One of the best experts on this subject based on the ideXlab platform.
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Alanine Dehydrogenase from soybean nodule bacteroids: purification and properties.
Archives of biochemistry and biophysics, 1993Co-Authors: M.t. Smith, David W. EmerichAbstract:Abstract Alanine Dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH + 4 , pyruvate, L-Alanine, and NAD were 86 μM, 8.9 mM, 0.49 mM, 1 mM and 200 μM, respectively. High concentrations of pyruvate, L-Alanine, or NH + 4 caused inhibition of activity with K i ′s of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible. s. Inc.
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Alanine Dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies.
Journal of Biological Chemistry, 1993Co-Authors: M.t. Smith, David W. EmerichAbstract:Abstract The kinetic mechanism of Alanine Dehydrogenase from soybean nodule bacteroids was studied by initial velocity experiments with or without product inhibitors, dead-end inhibitors, or alternate substrates. Without inhibitors, double-reciprocal plots of initial velocity experiments showed intersecting lines, indicating a sequential mechanism. These initial velocity experiments also revealed rapid-equilibrium ordered binding of NH4+ prior to pyruvate. When NAD was varied at changing-fixed concentrations of L-Alanine, a nonlinear, concave down double-reciprocal plot was obtained. Substrate inhibition by pyruvate or L-Alanine with cosubstrates varied was uncompetitive giving further support to an ordered mechanism. Product inhibition studies showed that both NAD and NADH and pyruvate and L-Alanine were competitive. This suggested a Theorell-Chance mechanism. When product inhibition by L-Alanine was studied with NH4+ varied in a series of experiments at increasing concentrations of pyruvate, the inhibition was eliminated, as expected for a Theorell-Chance mechanism. Furthermore, when NADH, NH4+, and pyruvate were varied simultaneously, maintaining their concentrations at a constant ratio to each other, an infinite Vmax was obtained. pH studies of the kinetic parameters indicated that NH4+, rather than NH3, was the true substrate that binds to a residue on the enzyme with a pK of 8.1. In conclusion, the kinetic mechanism at pH 8.5 was determined to be a Ter-Bi Theorell-Chance. In the amination direction, the substrates add in the order: NADH, NH4+, pyruvate, with NH4+ binding in rapid-equilibrium. In the reverse direction, NAD adds first, followed by L-Alanine.
Harold G. Monbouquette - One of the best experts on this subject based on the ideXlab platform.
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a novel archaeal Alanine Dehydrogenase homologous to ornithine cyclodeaminase and μ crystallin
Journal of Bacteriology, 2004Co-Authors: Imke Schroder, Alexander Vadas, Eric N Johnson, Sierin Lim, Harold G. MonbouquetteAbstract:A novel Alanine Dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-Alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-Alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.
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structure of Alanine Dehydrogenase from archaeoglobus active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin
Journal of Molecular Biology, 2004Co-Authors: David T. Gallagher, Imke Schroder, Harold G. Monbouquette, Marcia J. Holden, Hugh Robinson, Natasha SmithAbstract:The hyperthermophilic archaeon Archaeoglobus fulgidus contains an l-Ala Dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial Dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent Dehydrogenases. The gene (Genbank AAB89583; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other Dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.
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Room-temperature synthesis of L-Alanine using the Alanine Dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus.
Biotechnology progress, 2002Co-Authors: Alexander Vadas, Imke Schroder, Harold G. MonbouquetteAbstract:Alanine Dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-Alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate Dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-Alanine at room temperature was accompanied by no detectable loss of Alanine Dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.
M.t. Smith - One of the best experts on this subject based on the ideXlab platform.
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Alanine Dehydrogenase from soybean nodule bacteroids: purification and properties.
Archives of biochemistry and biophysics, 1993Co-Authors: M.t. Smith, David W. EmerichAbstract:Abstract Alanine Dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH + 4 , pyruvate, L-Alanine, and NAD were 86 μM, 8.9 mM, 0.49 mM, 1 mM and 200 μM, respectively. High concentrations of pyruvate, L-Alanine, or NH + 4 caused inhibition of activity with K i ′s of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible. s. Inc.
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Alanine Dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies.
Journal of Biological Chemistry, 1993Co-Authors: M.t. Smith, David W. EmerichAbstract:Abstract The kinetic mechanism of Alanine Dehydrogenase from soybean nodule bacteroids was studied by initial velocity experiments with or without product inhibitors, dead-end inhibitors, or alternate substrates. Without inhibitors, double-reciprocal plots of initial velocity experiments showed intersecting lines, indicating a sequential mechanism. These initial velocity experiments also revealed rapid-equilibrium ordered binding of NH4+ prior to pyruvate. When NAD was varied at changing-fixed concentrations of L-Alanine, a nonlinear, concave down double-reciprocal plot was obtained. Substrate inhibition by pyruvate or L-Alanine with cosubstrates varied was uncompetitive giving further support to an ordered mechanism. Product inhibition studies showed that both NAD and NADH and pyruvate and L-Alanine were competitive. This suggested a Theorell-Chance mechanism. When product inhibition by L-Alanine was studied with NH4+ varied in a series of experiments at increasing concentrations of pyruvate, the inhibition was eliminated, as expected for a Theorell-Chance mechanism. Furthermore, when NADH, NH4+, and pyruvate were varied simultaneously, maintaining their concentrations at a constant ratio to each other, an infinite Vmax was obtained. pH studies of the kinetic parameters indicated that NH4+, rather than NH3, was the true substrate that binds to a residue on the enzyme with a pK of 8.1. In conclusion, the kinetic mechanism at pH 8.5 was determined to be a Ter-Bi Theorell-Chance. In the amination direction, the substrates add in the order: NADH, NH4+, pyruvate, with NH4+ binding in rapid-equilibrium. In the reverse direction, NAD adds first, followed by L-Alanine.
Imke Schroder - One of the best experts on this subject based on the ideXlab platform.
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a novel archaeal Alanine Dehydrogenase homologous to ornithine cyclodeaminase and μ crystallin
Journal of Bacteriology, 2004Co-Authors: Imke Schroder, Alexander Vadas, Eric N Johnson, Sierin Lim, Harold G. MonbouquetteAbstract:A novel Alanine Dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of L-Alanine to pyruvate and NH4+. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The Km values for L-Alanine, NAD+, pyruvate, NADH, and NH4+ were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82 degrees C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25 degrees C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90 degrees C. At 25 degrees C in the presence of this salt solution, the enzyme was approximately 100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-mu-crystallin family of enzymes. Similar to the mu-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human mu-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.
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structure of Alanine Dehydrogenase from archaeoglobus active site analysis and relation to bacterial cyclodeaminases and mammalian mu crystallin
Journal of Molecular Biology, 2004Co-Authors: David T. Gallagher, Imke Schroder, Harold G. Monbouquette, Marcia J. Holden, Hugh Robinson, Natasha SmithAbstract:The hyperthermophilic archaeon Archaeoglobus fulgidus contains an l-Ala Dehydrogenase (AlaDH, EC 1.4.1.1) that is not homologous to known bacterial Dehydrogenases and appears to represent a previously unrecognized archaeal group of NAD-dependent Dehydrogenases. The gene (Genbank AAB89583; TIGR AF1665) was annotated initially as an ornithine cyclodeaminase (OCD) on the basis of strong homology with the mu crystallin/OCD protein family. We report the structure of the NAD-bound AF1665 AlaDH (AF-AlaDH) at 2.3 A in a C2 crystal form with the 70 kDa dimer in the asymmetric unit, as the first structural representative of this family. Consistent with its lack of homology to bacterial AlaDH proteins, which are mostly hexameric, the archaeal dimer has a novel structure. Although both types of AlaDH enzyme include a Rossmann-type NAD-binding domain, the arrangement of strands in the C-terminal half of this domain is novel, and the other (catalytic) domain in the archaeal protein has a new fold. The active site presents a cluster of conserved Arg and Lys side-chains over the pro-R face of the cofactor. In addition, the best ordered of the 338 water molecules in the structure is positioned well for mechanistic interaction. The overall structure and active site are compared with other Dehydrogenases, including the AlaDH from Phormidium lapideum. Implications for the catalytic mechanism and for the structures of homologs are considered. The archaeal AlaDH represents an ancient and previously undescribed subclass of Rossmann-fold proteins that includes bacterial ornithine and lysine cyclodeaminases, marsupial lens proteins and, in man, a thyroid hormone-binding protein that exhibits 30% sequence identity with AF1665.
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Room-temperature synthesis of L-Alanine using the Alanine Dehydrogenase of the hyperthermophilic archaeon Archaeoglobus fulgidus.
Biotechnology progress, 2002Co-Authors: Alexander Vadas, Imke Schroder, Harold G. MonbouquetteAbstract:Alanine Dehydrogenase from the hyperthermophilic archaeon Archaeoglobus fulgidus was used at room temperature for batch synthesis of L-Alanine by the reductive amination of pyruvate. The reaction mixture included yeast formate Dehydrogenase for regeneration of NADH with formate as electron donor. The synthesis of L-Alanine at room temperature was accompanied by no detectable loss of Alanine Dehydrogenase activity over 139 h and > or =99% consumption of pyruvate. The total number of enzyme turnovers was 5.1 million. This work demonstrates the potential utility of novel hyperthermostable enzymes that can be both very active and highly stable at moderate temperature.