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John J. Tanner - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline
'MDPI AG', 2017Co-Authors: David A. Korasick, Donald F. Becker, Benjamin W Arentson, Travis A Pemberton, John J. TannerAbstract:Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of l-Proline to l-glutamate using spatially separated Proline Dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde Dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by Proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with Proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that Proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of Proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that Proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that Proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to Proline binding in the GSAL site
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kinetic and structural characterization of tunnel perturbing mutants in bradyrhizobium japonicum Proline utilization a
Biochemistry, 2014Co-Authors: Benjamin W Arentson, John J. Tanner, Travis A Pemberton, Donald F. BeckerAbstract:Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of Proline to glutamate using fused Proline Dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate Dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutan...
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evidence for hysteretic substrate channeling in the Proline Dehydrogenase and δ1 pyrroline 5 carboxylate Dehydrogenase coupled reaction of Proline utilization a puta
Journal of Biological Chemistry, 2014Co-Authors: Michael A Moxley, John J. Tanner, Nikhilesh Sanyal, Navasona Krishnan, Donald F. BeckerAbstract:PutA (Proline utilization A) is a large bifunctional flavoenzyme with Proline Dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate Dehydrogenase (P5CDH) domains that catalyze the oxidation of l-Proline to l-glutamate in two successive reactions. In the PRODH active site, Proline undergoes a two-electron oxidation to Δ1-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ1-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD+-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of Proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.
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crystal structures and kinetics of monofunctional Proline Dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release
Biochemistry, 2012Co-Authors: Min Luo, Dhiraj Srivastava, Donald F. Becker, Benjamin W Arentson, John J. TannerAbstract:Proline Dehydrogenase catalyzes the FAD-dependent oxidation of Proline to Δ1- pyrroline-5-carboxylate, which is the first step of Proline catabolism. Here, we report the structures of Proline Dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the Proline analog L-tetrahydrofuroic acid and in the reduced state with the Proline site vacant. The analog binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by the α8 helix and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 A, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.
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crystal structure of the bifunctional Proline utilization a flavoenzyme from bradyrhizobium japonicum
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Dhiraj Srivastava, Donald F. Becker, Jonathan P Schuermann, Nikhilesh Sanyal, Navasona Krishnan, Tommi A White, Greg L Hura, Michael T Henzl, John J. TannerAbstract:The bifunctional Proline catabolic flavoenzyme, Proline utilization A (PutA), catalyzes the oxidation of Proline to glutamate via the sequential activities of FAD-dependent Proline Dehydrogenase (PRODH) and NAD+-dependent Δ1-pyrroline-5-carboxylate Dehydrogenase (P5CDH) domains. Although structures for some of the domains of PutA are known, a structure for the full-length protein has not previously been solved. Here we report the 2.1 Å resolution crystal structure of PutA from Bradyrhizobium japonicum, along with data from small-angle x-ray scattering, analytical ultracentrifugation, and steady-state and rapid-reaction kinetics. PutA forms a ring-shaped tetramer in solution having a diameter of 150 Å. Within each protomer, the PRODH and P5CDH active sites face each other at a distance of 41 Å and are connected by a large, irregularly shaped cavity. Kinetics measurements show that glutamate production occurs without a lag phase, suggesting that the intermediate, Δ1-pyrroline-5-carboxylate, is preferably transferred to the P5CDH domain rather than released into the bulk medium. The structural and kinetic data imply that the cavity serves both as a microscopic vessel for the hydrolysis of Δ1-pyrroline-5-carboxylate to glutamate semialdehyde and a protected conduit for the transport of glutamate semialdehyde to the P5CDH active site.
Donald F. Becker - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis for the Substrate Inhibition of Proline Utilization A by Proline
'MDPI AG', 2017Co-Authors: David A. Korasick, Donald F. Becker, Benjamin W Arentson, Travis A Pemberton, John J. TannerAbstract:Proline utilization A (PutA) is a bifunctional flavoenzyme that catalyzes the two-step oxidation of l-Proline to l-glutamate using spatially separated Proline Dehydrogenase (PRODH) and l-glutamate-γ-semialdehyde Dehydrogenase (GSALDH) active sites. Substrate inhibition of the coupled PRODH-GSALDH reaction by Proline is a common kinetic feature of PutAs, yet the structural basis for this phenomenon remains unknown. To understand the mechanism of substrate inhibition, we determined the 2.15 Å resolution crystal structure of Bradyrhizobium japonicum PutA complexed with Proline. Proline was discovered in five locations remote from the PRODH active site. Most notably, strong electron density indicated that Proline bound tightly to the GSAL binding site of the GSALDH active site. The pose and interactions of Proline bound in this site are remarkably similar to those of the natural aldehyde substrate, GSAL, implying that Proline inhibits the GSALDH reaction of PutA. Kinetic measurements show that Proline is a competitive inhibitor of the PutA GSALDH reaction. Together, the structural and kinetic data show that substrate inhibition of the PutA coupled reaction is due to Proline binding in the GSAL site
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kinetic and structural characterization of tunnel perturbing mutants in bradyrhizobium japonicum Proline utilization a
Biochemistry, 2014Co-Authors: Benjamin W Arentson, John J. Tanner, Travis A Pemberton, Donald F. BeckerAbstract:Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of Proline to glutamate using fused Proline Dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate Dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ1-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall rate of the PRODH–P5CDH channeling reaction. X-ray crystal structures of D779Y and D779W revealed that the large side chains caused a constriction in the central section of the tunnel, thus likely impeding the travel of P5C/GSA in the channel. The D779Y and D779W mutan...
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evidence for hysteretic substrate channeling in the Proline Dehydrogenase and δ1 pyrroline 5 carboxylate Dehydrogenase coupled reaction of Proline utilization a puta
Journal of Biological Chemistry, 2014Co-Authors: Michael A Moxley, John J. Tanner, Nikhilesh Sanyal, Navasona Krishnan, Donald F. BeckerAbstract:PutA (Proline utilization A) is a large bifunctional flavoenzyme with Proline Dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate Dehydrogenase (P5CDH) domains that catalyze the oxidation of l-Proline to l-glutamate in two successive reactions. In the PRODH active site, Proline undergoes a two-electron oxidation to Δ1-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ1-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD+-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of Proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.
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crystal structures and kinetics of monofunctional Proline Dehydrogenase provide insight into substrate recognition and conformational changes associated with flavin reduction and product release
Biochemistry, 2012Co-Authors: Min Luo, Dhiraj Srivastava, Donald F. Becker, Benjamin W Arentson, John J. TannerAbstract:Proline Dehydrogenase catalyzes the FAD-dependent oxidation of Proline to Δ1- pyrroline-5-carboxylate, which is the first step of Proline catabolism. Here, we report the structures of Proline Dehydrogenase from Deinococcus radiodurans in the oxidized state complexed with the Proline analog L-tetrahydrofuroic acid and in the reduced state with the Proline site vacant. The analog binds against the si face of the FAD isoalloxazine and is protected from bulk solvent by the α8 helix and the β1-α1 loop. The FAD ribityl chain adopts two conformations in the E-S complex, which is unprecedented for flavoenzymes. One of the conformations is novel for the PRODH superfamily and may contribute to the low substrate affinity of Deinococcus PRODH. Reduction of the crystalline enzyme-inhibitor complex causes profound structural changes, including 20° butterfly bending of the isoalloxazine, crankshaft rotation of the ribityl, shifting of α8 by 1.7 A, reconfiguration of the β1-α1 loop, and rupture of the Arg291-Glu64 ion pair. These changes dramatically open the active site to facilitate product release and allow electron acceptors access to the reduced flavin. The structures suggest that the ion pair, which is conserved in the PRODH superfamily, functions as the active site gate. Mutagenesis of Glu64 to Ala decreases catalytic efficiency 27-fold, which demonstrates the importance of the gate. Mutation of Gly63 decreases efficiency 140-fold, which suggests that flexibility of the β1-α1 loop is essential for optimal catalysis. The large conformational changes that are required to form the E-S complex suggest that conformational selection plays a role in substrate recognition.
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role of apoptosis inducing factor Proline Dehydrogenase and nadph oxidase in apoptosis and oxidative stress
Cell Health and Cytoskeleton, 2012Co-Authors: Sathish Kumar Natarajan, Donald F. BeckerAbstract:Flavoproteins catalyze a variety of reactions utilizing flavin mononucleotide or flavin adenine dinucleotide as cofactors. The oxidoreductase properties of flavoenzymes implicate them in redox homeostasis, oxidative stress, and various cellular processes, including programmed cell death. Here we explore three critical flavoproteins involved in apoptosis and redox signaling, ie, apoptosis-inducing factor (AIF), Proline Dehydrogenase, and NADPH oxidase. These proteins have diverse biochemical functions and influence apoptotic signaling by unique mechanisms. The role of AIF in apoptotic signaling is two-fold, with AIF changing intracellular location from the inner mitochondrial membrane space to the nucleus upon exposure of cells to apoptotic stimuli. In the mitochondria, AIF enhances mitochondrial bioenergetics and complex I activity/assembly to help maintain proper cellular redox homeostasis. After translocating to the nucleus, AIF forms a chromatin degrading complex with other proteins, such as cyclophilin A. AIF translocation from the mitochondria to the nucleus is triggered by oxidative stress, implicating AIF as a mitochondrial redox sensor. Proline Dehydrogenase is a membrane-associated flavoenzyme in the mitochondrion that catalyzes the rate-limiting step of Proline oxidation. Upregulation of Proline Dehydrogenase by the tumor suppressor, p53, leads to enhanced mitochondrial reactive oxygen species that induce the intrinsic apoptotic pathway. NADPH oxidases are a group of enzymes that generate reactive oxygen species for oxidative stress and signaling purposes. Upon activation, NADPH oxidase 2 generates a burst of superoxide in neutrophils that leads to killing of microbes during phagocytosis. NADPH oxidases also participate in redox signaling that involves hydrogen peroxide-mediated activation of different pathways regulating cell proliferation and cell death. Potential therapeutic strategies for each enzyme are also highlighted.
James M Phang - One of the best experts on this subject based on the ideXlab platform.
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co regulation of mitochondrial respiration by Proline Dehydrogenase oxidase and succinate
Amino Acids, 2016Co-Authors: Chad N Hancock, Wei Liu, Gregory W Alvord, James M PhangAbstract:Proline Dehydrogenase/oxidase (PRODH/POX) is a mitochondrial protein critical to multiple stress pathways. Because of the roles of PRODH/POX in signaling, and its shared localization to the mitochondrial inner membrane with the electron transport chain (ETC), we investigated whether there was a direct relationship between PRODH/POX and regulation of the ETC. We found that PRODH/POX binds directly to CoQ1 and that CoQ1-dependent PRODH/POX activity required functional Complex III and Complex IV. PRODH/POX supported respiration in living cells during nutrient stress; however, expression of PRODH/POX resulted in an overall decrease in respiratory fitness. Effects on respiratory fitness were inhibited by DHP and NAC, indicating that these effects were mediated by PRODH/POX-dependent reactive oxygen species (ROS) generation. PRODH/POX expression resulted in a dose-dependent down-regulation of Complexes I–IV of the ETC, and this effect was also mitigated by the addition of DHP and NAC. We found that succinate was an uncompetitive inhibitor of PRODH/POX activity, inhibited ROS generation by PRODH/POX, and alleviated PRODH/POX effects on respiratory fitness. The findings demonstrate novel cross-talk between Proline and succinate respiration in vivo and provide mechanistic insights into observations from previous animal studies. Our results suggest a potential regulatory loop between PRODH/POX and succinate in regulation of mitochondrial respiration.
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Proline biosynthesis augments tumor cell growth and aerobic glycolysis involvement of pyridine nucleotides
Scientific Reports, 2015Co-Authors: Chad N Hancock, Joseph W Fischer, Meredith Harman, James M PhangAbstract:The metabolism of the nonessential amino acid Proline contributes to tumor metabolic reprogramming. Previously we showed that MYC increases Proline biosynthesis (PB) from glutamine. Here we show MYC increases the expression of the enzymes in PB at both protein and mRNA levels. Blockade of PB decreases tumor cell growth and energy production. Addition of Δ(1)-pyrroline-5-carboxylate (P5C) or Proline reverses the effects of P5C synthase knockdown but not P5C reductases knockdown. Importantly, the reversal effect of Proline was blocked by concomitant Proline Dehydrogenase/oxidase (PRODH/POX) knockdown. These findings suggest that the important regulatory contribution of PB to tumor growth derives from metabolic cycling between Proline and P5C rather than product Proline or intermediate P5C. We further document the critical role of PB in maintaining pyridine nucleotide levels by connecting the Proline cycle to glycolysis and to the oxidative arm of the pentose phosphate pathway. These findings establish a novel function of PB in tumorigenesis, linking the reprogramming of glucose, glutamine and pyridine nucleotides, and may provide a novel target for antitumor therapy.
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Proline Dehydrogenase oxidase in cancer
Biofactors, 2012Co-Authors: Wei Liu, James M PhangAbstract:Proline Dehydrogenase (oxidase, PRODH/POX), the first enzyme in the Proline degradative pathway, plays a special role in tumorigenesis and tumor development. Proline metabolism catalyzed by PRODH/POX is closely linked with the tricarboxylic acid (TCA) cycle and urea cycle. The Proline cycle formed by the interconversion of Proline and Δ(1) -pyrroline-5-carboxylate (P5C) between mitochondria and cytosol interlocks with pentose phosphate pathway. Importantly, by catalyzing Proline to P5C, PRODH/POX donates electrons into the electron transport chain to generate ROS or ATP. In earlier studies, we found that PRODH/POX functions as a tumor suppressor to initiate apoptosis, inhibit tumor growth, and block the cell cycle, all by ROS signaling. It also suppresses hypoxia inducible factor signaling by increasing α-ketoglutarate. During tumor progression, PRODH/POX is under the control of various tumor-associated factors, such as tumor suppressor p53, inflammatory factor peroxisome proliferator-activated receptor gamma (PPARγ), onco-miRNA miR-23b*, and oncogenic transcription factor c-MYC. Recent studies revealed the two-sided features of PRODH/POX-mediated regulation. Under metabolic stress such as oxygen and glucose deprivation, PRODH/POX can be induced to serve as a tumor survival factor through ATP production or ROS-induced autophagy. The paradoxical roles of PRODH/POX can be understood considering the temporal and spatial context of the tumor. Further studies will provide additional insights into this protein and on its metabolic effects in tumors, which may lead to new therapeutic strategies.
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Proline Dehydrogenase oxidase a mitochondrial tumor suppressor and autophagy under the hypoxia microenvironment
Autophagy, 2012Co-Authors: Wei Liu, James M PhangAbstract:Proline Dehydrogenase (oxidase, PRODH/POX), the first enzyme in the pathway of Proline catabolism, has been identified as a mitochondrial, metabolic tumor suppressor, which is downregulated in a variety of human tumors. However, our recent findings show that PRODH/POX is upregulated by hypoxia in vitro and in vivo. The combination of low glucose and hypoxia produces additive effects on PRODH/POX expression. Both hypoxia and glucose depletion enhance PRODH/POX expression through AMP-activated protein kinase (AMPK) activation to promote tumor cell survival. Nevertheless, the mechanisms underlying PRODH/POX prosurvival functions are different for hypoxia and low-glucose conditions. Glucose depletion with or without hypoxia elevates PRODH/POX and Proline utilization to supply ATP for cellular energy needs. Interestingly, under hypoxia PRODH/POX induces protective autophagy by generating reactive oxygen species (ROS). AMPK is the main initiator of stress-triggered autophagy. Thus, PRODH/POX acts as a downstream effector of AMPK in the activation of autophagy under hypoxia. This regulation was confirmed to be independent of the mechanistic target of rapamycin (MTOR) pathway, a major downstream target of AMPK signaling.
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the metabolism of Proline a stress substrate modulates carcinogenic pathways
Amino Acids, 2008Co-Authors: James M Phang, Steven P Donald, Jui Pandhare, Yongmin LiuAbstract:The resurgence of interest in tumor metabolism has led investigators to emphasize the metabolism of Proline as a “stress substrate” and to suggest this pathway as a potential anti-tumor target. Proline oxidase, a.k.a. Proline Dehydrogenase (POX/PRODH), catalyzes the first step in Proline degradation and uses Proline to generate ATP for survival or reactive oxygen species for programmed cell death. POX/PRODH is induced by p53 under genotoxic stress and initiates apoptosis by both mitochondrial and death receptor pathways. Furthermore, POX/PRODH is induced by PPARγ and its pharmacologic ligands, the thiazolidinediones. The anti-tumor effects of PPARγ may be critically dependent on POX/PRODH. In addition, it is upregulated by nutrient stress through the mTOR pathway to maintain ATP levels. We propose that Proline is made available as a stress substrate by the degradation of collagen in the microenvironmental extracellular matrix by matrix metalloproteinases. In a manner analogous to autophagy, this Proline-dependent process for bioenergetics from collagen in extracellular matrix can be designated “ecophagy”.
Jerzy Palka - One of the best experts on this subject based on the ideXlab platform.
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understanding the role of key amino acids in regulation of Proline Dehydrogenase Proline oxidase prodh pox dependent apoptosis autophagy as an approach to targeted cancer therapy
Molecular and Cellular Biochemistry, 2020Co-Authors: Thi Yen Ly Huynh, Ilona Zareba, Weronika Baszanowska, Sylwia Lewoniewska, Jerzy PalkaAbstract:In stress conditions, as neoplastic transformation, amino acids serve not only as nutrients to maintain the cell survival but also as mediators of several regulatory pathways which are involved in apoptosis and autophagy. Especially, under glucose deprivation, in order to maintain the cell survival, Proline and glutamine together with other glutamine-derived products such as glutamate, alpha-ketoglutarate, and ornithine serve as alternative sources of energy. They are substrates for production of pyrroline-5-carboxylate which is the product of conversion of Proline by Proline Dehydrogenase/ Proline oxidase (PRODH/POX) to produce ATP for protective autophagy or reactive oxygen species for apoptosis. Interconversion of Proline, ornithine, and glutamate may therefore regulate PRODH/POX-dependent apoptosis/autophagy. The key amino acid is Proline, circulating between mitochondria and cytoplasm in the Proline cycle. This shuttle is known as Proline cycle. It is coupled to pentose phosphate pathway producing nucleotides for DNA biosynthesis. PRODH/POX is also linked to p53 and AMP-activated protein kinase (AMPK)-dependent pathways. Proline availability for PRODH/POX-dependent apoptosis/autophagy is regulated at the level of collagen biosynthesis (Proline utilizing process) and prolidase activity (Proline supporting process). In this review, we suggest that amino acid metabolism linking TCA and Urea cycles affect PRODH/POX-dependent apoptosis/autophagy and the knowledge might be useful to targeted cancer therapy.
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prolidase Proline Dehydrogenase Proline oxidase collagen biosynthesis axis as a potential interface of apoptosis autophagy
Biofactors, 2016Co-Authors: Ilona Zareba, Jerzy PalkaAbstract:Prolidase is a cytosolic imidodipeptidase that specifically splits imidodipeptides with C-terminal Proline or hydroxyProline. The enzyme plays an important role in the recycling of Proline from imidodipeptides for resynthesis of collagen and other Proline-containing proteins. The mechanism of prolidase-dependent regulation of collagen biosynthesis was found at both transcriptional and post-transcriptional level. The increase in the enzyme activity is due to its phosphorylation on serine/threonine residues. Prolidase-dependent transcriptional regulation of collagen biosynthesis was found at the level of NF-κB, known inhibitor of type I collagen gene expression. Proline Dehydrogenase/Proline oxidase (PRODH/POX) is flavin-dependent enzyme associated with the inner mitochondrial membrane. The enzyme catalyzes conversion of Proline into Δ(1) -pyrroline-5-carboxylate (P5C), during which reactive oxygen species (ROS) are produced, inducing intrinsic and extrinsic apoptotic pathways. Alternatively, under low glucose stress, PRODH/POX activation produces ATP for energy supply and survival. Of special interest is that PRODH/POX gene is induced by P53 and peroxisome proliferator-activated gamma receptor (PPARγ). Among down-regulators of PRODH/POX is an oncogenic transcription factor c-MYC and miR-23b*. On the other hand, PRODH/POX suppresses HIF-1α transcriptional activity, the MAPK pathway, cyclooxygenase-2, epidermal growth factor receptor and Wnt/b-catenin signaling. PRODH/POX expression is often down-regulated in various tumors, limiting mitochondrial Proline utilization to P5C. It is accompanied by increased cytoplasmic level of Proline. Proline availability for PRODH/POX-dependent ATP or ROS generation depends on activity of prolidase and utilization of Proline in process of collagen biosynthesis. Therefore, Prolidase-PRODH/POX-Collagen Biosynthesis axis may represent potential interface that regulate apoptosis and survival. © 2016 BioFactors, 42(4):341-348, 2016.
Toshihisa Ohshima - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of novel dye linked l Proline Dehydrogenase from hyperthermophilic archaeon aeropyrum pernix
Journal of Biological Chemistry, 2012Co-Authors: Haruhiko Sakuraba, Ryushi Kawakami, Takenori Satomura, Yusuke Hara, Kwang Kim, Kazunari Yoneda, Toshihisa OhshimaAbstract:Two types of dye-linked l-Proline Dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of l-Proline Dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied l-Proline Dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 β-subunit, which is responsible for catalyzing l-Proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu428, which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an l-Proline molecule was entrapped within the active site cavity. The Km value of a Leu428 deletion mutant for l-Proline was about 800 times larger than the Km value of the wild-type enzyme, although the kcat values did not differ much between the two enzymes. This suggests the C-terminal Leu428 is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with l-Proline bound, and it provides new insight into the substrate binding of LPDH.
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gene expression and characterization of a third type of dye linked l Proline Dehydrogenase from the aerobic hyperthermophilic archaeon aeropyrum pernix
Bioscience Biotechnology and Biochemistry, 2012Co-Authors: Takenori Satomura, Haruhiko Sakuraba, Shinichiro Suye, Yusuke Hara, Toshihisa OhshimaAbstract:A third novel type of dye-linked L-Proline Dehydrogenase (LPDH) has recently been found in the hyperthermophilic archaeon, Pyrobaculum calidifontis, by Satomura et al. The gene encoding the enzyme homologue was identified in the aerobic hyperthermophilic archaeon, Aeropyrum pernix. The gene was successfully expressed in Escherichia coli, and the product was purified to homogeneity and characterized. The expressed enzyme was highly thermostable LPDH having a molecular mass of about 88 kDa and a homodimeric structure. The preferred substrate for the enzyme was L-Proline with 2,6-dichloroindophenol (DCIP) as the electron acceptor. However, the enzyme did not utilize ferricyanide as the electron acceptor, in contrast to all other known LPDHs. The electrochemical determination of L-Proline at concentrations from 0 to 0.7 mM was achieved by using A. pernix LPDH. A phylogenetic analysis revealed A. pernix LPDH to be clustered with the third type of LPDHs, and to be clearly separated from the clusters of previous...
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crystallization and preliminary x ray analysis of a dye linked d lactate Dehydrogenase from the aerobic hyperthermophilic archaeon aeropyrum pernix
Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2010Co-Authors: Takenori Shibahara, Ryushi Kawakami, Toshihisa Ohshima, Takenori Satomura, Haruhiko SakurabaAbstract:A novel dye-linked l-Proline Dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix was crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol 8000 as the precipitant. The crystals belonged to the tetragonal space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 61.1, c = 276.3 A, and diffracted to 2.87 A resolution using a Cu Kα rotating-anode generator with an R-AXIS VII detector. The asymmetric unit contained one protein molecule, giving a crystal volume per enzyme mass (V M) of 2.75 A3 Da−1 and a solvent content of 55.3%.
