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1.
Semin Dial ; 30(3): 187-192, 2017 05.
Article in English | MEDLINE | ID: mdl-28229483

ABSTRACT

End-stage renal disease is frequently accompanied by cardiac comorbidity that warrants treatment with a cardiovascular implantable electronic device (permanent pacemaker or implantable cardioverter-defibrillator). In the United States, chronic hemodialysis (HD) population, cardiac implantable devices are present in up to 10.5% of patients; a venous HD catheter is utilized for blood access in 18% of prevalent patients. The concomitant presence of a venous HD catheter and cardiovascular implantable device creates a high-risk circumstance, with potential for causing symptomatic central venous stenosis, and for developing complicated endovascular infection. This dangerous combination may be avoided for many patients by utilizing nondialysis methods for management of advanced chronic kidney disease, initiating dialysis without venous catheter access, or managing cardiac rhythm disorders without use of transvenous cardiac implantable electronic devices. In those situations where the combination of a venous HD catheter and cardiac implantable device is unavoidable, there are strategies to minimize duration of venous catheter access, and to reduce risks for infectious complications. It is essential for nephrologists and cardiologists to understand the indications, alternatives, and risks involved with venous HD access and cardiac implantable devices. Coordinated management of renal disease and cardiac rhythm disorders has potential to minimize risks, improve outcomes, and substantially reduce the cost of care.


Subject(s)
Arrhythmias, Cardiac/therapy , Catheters, Indwelling , Defibrillators, Implantable , Kidney Failure, Chronic/therapy , Pacemaker, Artificial , Renal Dialysis/instrumentation , Risk Assessment , Arrhythmias, Cardiac/complications , Arrhythmias, Cardiac/epidemiology , Humans , Incidence , Kidney Failure, Chronic/complications , Kidney Failure, Chronic/epidemiology , Prognosis , Risk Factors , United States/epidemiology
4.
Biochim Biophys Acta ; 1830(6): 3391-8, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23454351

ABSTRACT

BACKGROUND: In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol. METHODS: Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol. RESULTS: In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol. CONCLUSION: This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol. GENERAL SIGNIFICANCE: The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Ethanol/metabolism , Isoenzymes/metabolism , Mitochondrial Proteins/metabolism , Retinal Dehydrogenase/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Aldehyde Dehydrogenase, Mitochondrial , Cytosol/enzymology , Gene Deletion , Genetic Complementation Test , Humans , Isoenzymes/genetics , Mitochondria/enzymology , Mitochondria/genetics , Mitochondrial Proteins/genetics , Retinal Dehydrogenase/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
5.
Chem Biol Interact ; 202(1-3): 32-40, 2013 Feb 25.
Article in English | MEDLINE | ID: mdl-23295226

ABSTRACT

Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)(+) coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Threonine/metabolism , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase, Mitochondrial , Amino Acid Substitution , Catalysis , Catalytic Domain , Coenzymes/genetics , Coenzymes/metabolism , Humans , Kinetics , NAD/genetics , NAD/metabolism , Niacinamide/genetics , Niacinamide/metabolism , Protein Binding , Protein Structure, Secondary , Threonine/genetics
6.
Plant J ; 59(2): 256-65, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19292760

ABSTRACT

Benzoic acid (BA) is an important building block in a wide spectrum of compounds varying from primary metabolites to secondary products. Benzoic acid biosynthesis from L-phenylalanine requires shortening of the propyl side chain by two carbons, which can occur via a beta-oxidative pathway or a non-beta-oxidative pathway, with benzaldehyde as a key intermediate. The non-beta-oxidative route requires benzaldehyde dehydrogenase (BALDH) to convert benzaldehyde to BA. Using a functional genomic approach, we identified an Antirrhinum majus (snapdragon) BALDH, which exhibits 40% identity to bacterial BALDH. Transcript profiling, biochemical characterization of the purified recombinant protein, molecular homology modeling, in vivo stable isotope labeling, and transient expression in petunia flowers reveal that BALDH is capable of oxidizing benzaldehyde to BA in vivo. GFP localization and immunogold labeling studies show that this biochemical step occurs in the mitochondria, raising a question about the role of subcellular compartmentalization in BA biosynthesis.


Subject(s)
Antirrhinum/enzymology , Benzaldehyde Dehydrogenase (NADP+)/metabolism , Benzoic Acid/metabolism , Plant Proteins/metabolism , Antirrhinum/genetics , Benzaldehyde Dehydrogenase (NADP+)/genetics , DNA, Complementary/genetics , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Petunia/genetics , Petunia/metabolism , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , RNA, Plant/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
8.
Biochem Pharmacol ; 76(5): 690-6, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18647600

ABSTRACT

Cyclophosphamides are pro-drugs whose killing agent is produced from an aldehyde that is formed by the action of a P450 oxidation step. The mustard from the aldehyde can destroy bone marrow cells as well as the tumor. Aldehyde dehydrogenase (EC 1.2.1.3) can oxidize the aldehyde and hence inactivate the cytotoxic intermediate but bone marrow has little, if any, of the enzyme. Others have shown that over-expression of the enzyme can afford protection of the marrow. A T186S mutant of the human stomach enzyme (ALDH3) that we developed has increased activity against the aldehyde compared to the native enzyme and HeLa cells transformed with the point mutant are better protected against the killing effect of the drug. It took threefold more drug to kill 90% of the cells transformed with the mutant compared to the native enzyme (15.8 compared to 5.1mM of a precursor of the toxic aldehyde). Analysis of molecular models makes it appear that removing the methyl group of threonine in the T186S mutant allows the bulky aldehyde to bind better. The mutant was found to be a poorer enzyme when small substrates such as benzaldehyde derivatives were investigated. Thus, the enzyme appears to be better only with large substrates such as the one produced by cyclophosphamide.


Subject(s)
Aldehyde Dehydrogenase/genetics , Antineoplastic Agents, Alkylating/toxicity , Cyclophosphamide/toxicity , Cytoprotection/genetics , Point Mutation , Prodrugs/toxicity , Aldehyde Dehydrogenase/metabolism , Aldehydes/metabolism , Blotting, Western , Bone Marrow Cells/drug effects , Bone Marrow Cells/enzymology , Cloning, Molecular , HeLa Cells , Humans , Phosphoramide Mustards/metabolism , Stomach/enzymology , Transfection
9.
FASEB J ; 22(7): 2561-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18272654

ABSTRACT

Recent studies suggest that the mitochondrial aldehyde dehydrogenase (ALDH)2 is involved in vascular bioactivation of nitroglycerin (GTN). However, neither expression of ALDH2 nor its functional role in GTN bioactivation has been reported for the main drug target in humans, namely capacitance vessels. We investigated whether ALDH2 is expressed in human veins and whether inhibition of the enzyme attenuates nitroglycerin effects in these vessels. We determined expression of ALDH2 and dehydrogenase activity in human veins by reverse transcriptase-polymerase chain reaction, Western blotting, and immunofluorescence microscopy. In vitro contraction experiments were performed in the presence or absence of the ALDH inhibitors chloral hydrate, cyanamide, and ethoxycyclopropanol. Concentration response curves were determined for the alpha-agonist phenylephrine, nitroglycerin, and the direct NO donor diethylamine NONOate (DEA-NONOate). ALDH2 expression was largely confined to smooth muscle cells as determined by confocal immunofluorescence microscopy. Contractile responses to phenylephrine were unaffected by all ALDH inhibitors tested. In clear contrast, the ALDH inhibitors significantly reduced the potency of nitroglycerin by approximately 1 order of magnitude (P < or = 0.01). Neither of the inhibitors affected the potency of the direct NO donor DEA-NONOate, which ruled out nonspecific effects on the NO signaling cascade. In human capacitance vessels, ALDH2 is a key enzyme in the biotransformation of the frequently used antianginal drug nitroglycerin.


Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Nitroglycerin/pharmacology , Vasodilation/physiology , Veins/enzymology , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase, Mitochondrial , Chloral Hydrate/pharmacology , Cyanamide/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Kinetics , Mitochondria/drug effects , Mitochondria/enzymology , Reverse Transcriptase Polymerase Chain Reaction , Vasodilation/drug effects , Vasodilator Agents/pharmacology , Veins/cytology , Veins/drug effects , Veins/physiology
10.
J Am Coll Cardiol ; 50(23): 2226-32, 2007 Dec 04.
Article in English | MEDLINE | ID: mdl-18061070

ABSTRACT

OBJECTIVES: We tested the hypothesis of whether an inhibition of the nitroglycerin (GTN) bioactivating enzyme mitochondrial aldehyde dehydrogenase (ALDH-2) contributes to GTN tolerance in human blood vessels. BACKGROUND: The hemodynamic effects of GTN are rapidly blunted by the development of tolerance, a phenomenon associated with increased formation of reactive oxygen species (ROS). Recent studies suggest that ROS-induced inhibition of ALDH-2 accounts for tolerance in animal models. METHODS: Segments of surgically removed arteria mammaria and vena saphena from patients undergoing coronary bypass surgery were used to examine the vascular responsiveness to GTN and the endothelium-dependent vasodilator acetylcholine. The ALDH-2 activity and expression in these segments were assessed by the conversion of a benzaldehyde or its derivative to the benzoic acid metabolite and by Western blotting technique. RESULTS: In contrast to patients not treated with nitrates (n = 36), patients treated with GTN for 48 h (n = 14) before surgery showed tolerance to GTN and endothelial dysfunction in arterial and venous vessels. In vivo GTN tolerance was mimicked in vitro by incubation of nontolerant vessels with the ALDH-2 inhibitor benomyl. In vivo GTN treatment decreased vascular aldehyde dehydrogenase activity compared with nontolerant vessels and decreased the expression of ALDH-2 in arterial tissue. Incubation of control venous vessels with GTN caused a significant attenuation of aldehyde dehydrogenase activity that was reversed by presence of the sulfhydryl group donor dithiothreitol. CONCLUSIONS: Long-term GTN treatment induces tolerance and endothelial dysfunction in human vessels, associated with an inhibition and down-regulation of vascular ALDH-2. Thus, these findings extend results of previous animal studies to humans.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Drug Tolerance/physiology , Mammary Arteries/drug effects , Myocardial Infarction/enzymology , Nitroglycerin/pharmacology , Saphenous Vein/drug effects , Acetylcholine/pharmacology , Aged , Aldehyde Dehydrogenase, Mitochondrial , Drug Administration Schedule , Female , Humans , Male , Mammary Arteries/enzymology , Mammary Arteries/physiopathology , Myocardial Infarction/physiopathology , Myocardial Infarction/therapy , Nitric Oxide Synthase Type III/metabolism , Nitroglycerin/administration & dosage , Oxidative Stress/physiology , Saphenous Vein/enzymology , Saphenous Vein/physiopathology , Tissue Culture Techniques , Vasodilator Agents/pharmacology
11.
J Biol Chem ; 282(51): 37266-75, 2007 Dec 21.
Article in English | MEDLINE | ID: mdl-17959599

ABSTRACT

It is not known why leader peptides are removed by the mitochondrial processing peptidase after import into the matrix space. The leaders of yeast aldehyde dehydrogenase (pALDH) and malate dehydrogenase were mutated so that they would not be processed after import. The recombinant nonprocessed precursor of yeast pALDH possessed a similar specific activity as the corresponding mature form but was much less stable. The nonprocessed pALDH was transformed into a yeast strain missing ALDHs. The transformed yeast grew slowly on ethanol as the sole carbon source showing that the nonprocessed precursor was functional in vivo. Western blot analysis showed that the amount of precursor was 15-20% of that found in cells transformed with the native enzyme. Pulse-chase experiments revealed that the turnover rate for the nonprocessed precursor was greater than that of the mature protein indicating that the nonprocessed precursor could have been degraded. By using carbonyl cyanide m-chlorophenylhydrazone, we showed that the nonprocessed precursor was degraded in the matrix space. The nonprocessed precursor forms of precursor yeast malate dehydrogenase and rat liver pALDH also were degraded in the matrix space of HeLa cell mitochondria faster than their corresponding mature forms. In the presence of o-phenanthroline, an inhibitor of mitochondrial processing peptidase, the wild type precursor was readily degraded in the matrix space. Collectively, this study showed that the precursor form is less stable in the matrix space than is the mature form and provides an explanation for why the leader peptide is removed from the precursors.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Malate Dehydrogenase/metabolism , Metalloendopeptidases/metabolism , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Protein Precursors/metabolism , Protein Sorting Signals/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Aldehyde Dehydrogenase/genetics , Animals , HeLa Cells , Humans , Liver/enzymology , Malate Dehydrogenase/genetics , Metalloendopeptidases/genetics , Mitochondria/genetics , Mitochondrial Proteins/genetics , Protein Precursors/genetics , Protein Processing, Post-Translational , Protein Transport/physiology , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Mitochondrial Processing Peptidase
12.
Adv Drug Deliv Rev ; 59(8): 729-38, 2007 Aug 10.
Article in English | MEDLINE | ID: mdl-17659805

ABSTRACT

Mitochondria is where the bulk of the cell's ATP is produced. Mutations occur to genes coding for members of the complexes involved in energy production. Some are a result of damages to nuclear coded genes and others to mitochondrial coded genes. This review describes approaches to bring small molecules, proteins and RNA/DNA into mitochondria. The purpose is to repair damaged genes as well as to interrupt mitochondrial function including energy production, oxygen radical formation and the apoptotic pathway.


Subject(s)
Drug Delivery Systems/methods , Macromolecular Substances/administration & dosage , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Peptide Nucleic Acids/pharmacokinetics , Antioxidants/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Carrier Proteins/metabolism , DNA Repair/drug effects , DNA Repair/genetics , DNA, Mitochondrial/drug effects , DNA, Mitochondrial/genetics , Humans , Macromolecular Substances/metabolism , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Onium Compounds/metabolism , Oxidative Stress , Peptide Nucleic Acids/metabolism , Protein Sorting Signals/genetics , Reactive Oxygen Species/metabolism , Saccharomyces cerevisiae , Trityl Compounds/metabolism
13.
Arterioscler Thromb Vasc Biol ; 27(8): 1729-35, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17541025

ABSTRACT

OBJECTIVE: Nitrate tolerance is likely attributable to an increased production of reactive oxygen species (ROS) leading to an inhibition of the mitochondrial aldehyde dehydrogenase (ALDH-2), representing the nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN) bioactivating enzyme, and to impaired nitric oxide bioactivity and signaling. We tested whether differences in their capacity to induce heme oxygenase-1 (HO-1) might explain why PETN and not GTN therapy is devoid of nitrate and cross-tolerance. METHODS AND RESULTS: Wistar rats were treated with PETN or GTN (10.5 or 6.6 microg/kg/min for 4 days). In contrast to GTN, PETN did not induce nitrate tolerance or cross-tolerance as assessed by isometric tension recordings in isolated aortic rings. Vascular protein and mRNA expression of HO-1 and ferritin were increased in response to PETN but not GTN. In contrast to GTN therapy, NO signaling, ROS formation, and the activity of ALDH-2 (as assessed by an high-performance liquid chromatography-based method) were not significantly influenced by PETN. Inhibition of HO-1 expression by apigenin induced "tolerance" to PETN whereas HO-1 gene induction by hemin prevented tolerance in GTN treated rats. CONCLUSIONS: HO-1 expression and activity appear to play a key role in the development of nitrate tolerance and might represent an intrinsic antioxidative mechanism of therapeutic interest.


Subject(s)
Drug Tolerance , Heme Oxygenase-1/metabolism , Nitroglycerin/pharmacology , Pentaerythritol Tetranitrate/pharmacology , Aldehyde Dehydrogenase/metabolism , Animals , Chromatography, High Pressure Liquid , Cyclic GMP/metabolism , Disease Models, Animal , Endothelium, Vascular/drug effects , Free Radical Scavengers , Heme Oxygenase-1/drug effects , Male , Nitroglycerin/metabolism , Pentaerythritol Tetranitrate/metabolism , Probability , Random Allocation , Rats , Rats, Wistar , Reactive Oxygen Species , Reference Values , Sensitivity and Specificity
14.
Chem Res Toxicol ; 20(6): 887-95, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17480102

ABSTRACT

trans-4-Hydroxy-2-nonenal (HNE) is a cytotoxic alpha,beta-unsaturated aldehyde implicated in the pathology of multiple diseases involving oxidative damage. Oxidation of HNE by aldehyde dehydrogenases (ALDHs) to trans-4-hydroxy-2-nonenoic acid (HNEA) is a major route of metabolism in many organisms. HNE exists as two enantiomers, (R)-HNE and (S)-HNE, and in intact rat brain mitochondria, (R)-HNE is enantioselectively oxidized to HNEA. In this work, we further elucidated the basis of the enantioselective oxidation of HNE by brain mitochondria. Our results showed that (R)-HNE is oxidized enantioselectively by brain mitochondrial lysates with retention of stereoconfiguration of the C4 hydroxyl group. Purified rat ALDH5A enantioselectively oxidized (R)-HNE, whereas rat ALDH2 was not enantioselective. Kinetic data using (R)-HNE, (S)-HNE, and trans-2-nonenal in combination with computer-based modeling of ALDH5A suggest that the selectivity of (R)-HNE oxidation by ALDH5A is the result of the carbonyl carbon of (R)-HNE forming a more favorable Bürgi-Duntiz angle with the active site cysteine 293. The presence of Mg2+ ions altered the enantioselectivity of ALDH5A and ALDH2. Mg2+ ions suppressed (R)-HNE oxidation by ALDH5A to a greater extent than that of (S)-HNE. However, Mg2+ ions stimulated the enantioselective oxidation of (R)-HNE by ALDH2 while suppressing (S)-HNE oxidation. These results demonstrate that enantioselective utilization of substrates, including HNE, by ALDHs is dependent upon the ALDH isozyme and the presence of Mg 2+ ions.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Aldehydes/metabolism , Magnesium/pharmacology , Acetaldehyde/chemistry , Acetaldehyde/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehydes/chemistry , Animals , Catalysis/drug effects , Cations, Divalent/chemistry , Cations, Divalent/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Isoenzymes/chemistry , Isoenzymes/metabolism , Kinetics , Magnesium/chemistry , Models, Molecular , NAD/chemistry , NAD/metabolism , Oxidation-Reduction , Protein Conformation , Rats , Rats, Sprague-Dawley , Stereoisomerism , gamma-Aminobutyric Acid/analogs & derivatives , gamma-Aminobutyric Acid/chemistry , gamma-Aminobutyric Acid/metabolism
15.
J Biol Chem ; 282(17): 12940-50, 2007 Apr 27.
Article in English | MEDLINE | ID: mdl-17327228

ABSTRACT

The common mitochondrial aldehyde dehydrogenase (ALDH2) ALDH2(*)2 polymorphism is associated with impaired ethanol metabolism and decreased efficacy of nitroglycerin treatment. These physiological effects are due to the substitution of Lys for Glu-487 that reduces the k(cat) for these processes and increases the K(m) for NAD(+), as compared with ALDH2. In this study, we sought to understand the nature of the interactions that give rise to the loss of structural integrity and low activity in ALDH2(*)2 even when complexed with coenzyme. Consequently, we have solved the crystal structure of ALDH2(*)2 complexed with coenzyme to 2.5A(.) We have also solved the structures of a mutated form of ALDH2 where Arg-475 is replaced by Gln (R475Q). The structural and functional properties of the R475Q enzyme are intermediate between those of wild-type and the ALDH2(*)2 enzymes. In both cases, the binding of coenzyme restores most of the structural deficits observed in the apoenzyme structures. The binding of coenzyme to the R475Q enzyme restores its structure and catalytic properties to near wild-type levels. In contrast, the disordered helix within the coenzyme binding pocket of ALDH2(*)2 is reordered, but the active site is only partially reordered. Consistent with the structural data, ALDH2(*)2 showed a concentration-dependent increase in esterase activity and nitroglycerin reductase activity upon addition of coenzyme, but the levels of activity do not approach those of the wild-type enzyme or that of the R475Q enzyme. The data presented shows that Glu-487 maintains a critical function in linking the structure of the coenzyme-binding site to that of the active site through its interactions with Arg-264 and Arg-475, and in doing so, creates the stable structural scaffold conducive to catalysis.


Subject(s)
Aldehyde Dehydrogenase/chemistry , Coenzymes/chemistry , Mitochondrial Proteins/chemistry , Mutation, Missense , Oxidoreductases/chemistry , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Aldehyde Dehydrogenase, Mitochondrial , Amino Acid Substitution , Asian People , Catalysis , Coenzymes/metabolism , Crystallography, X-Ray , Ethanol/metabolism , Humans , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Models, Molecular , Nitroglycerin/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity Relationship
16.
J Biol Chem ; 282(1): 792-9, 2007 Jan 05.
Article in English | MEDLINE | ID: mdl-17102135

ABSTRACT

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of isolated aortic rings in response to nitroglycerin. Chronic nitroglycerin infusion lead to impaired vascular responses to nitroglycerin and decreased dehydrogenase activity, which was corrected by dihydrolipoic acid co-incubation. Superoxide, peroxynitrite, and nitroglycerin itself were highly efficient in inhibiting mitochondrial and yeast aldehyde dehydrogenase activity, which was restored by dithiol compounds such as dihydrolipoic acid and dithiothreitol. Hydrogen peroxide and nitric oxide were rather insensitive inhibitors. Our observations indicate that mitochondrial oxidative stress (especially superoxide and peroxynitrite) in response to organic nitrate treatment may inactivate aldehyde dehydrogenase thereby leading to nitrate tolerance. Glutathionylation obviously amplifies oxidative inactivation of the enzyme providing another regulatory pathway. Furthermore, the present data demonstrate that the mitochondrial dithiol compound dihydrolipoic acid restores mitochondrial aldehyde dehydrogenase activity via reduction of a disulfide at the active site and thereby improves nitrate tolerance.


Subject(s)
Aldehyde Dehydrogenase/physiology , Mitochondria/metabolism , Mitochondrial Proteins/physiology , Nitrates/chemistry , Oxidation-Reduction , Oxidative Stress , Thioctic Acid/chemistry , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase, Mitochondrial , Animals , Glutathione/chemistry , Glutathione/metabolism , Inhibitory Concentration 50 , Male , Mitochondrial Proteins/chemistry , Models, Biological , Myocardium/metabolism , Oxidants/chemistry , Oxidants/metabolism , Rats , Rats, Wistar
17.
Protein Sci ; 15(12): 2739-48, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17088320

ABSTRACT

Previous studies pointed to the importance of leucine residues in the binding of mitochondrial leader sequences to Tom20, an outer membrane protein translocator that initially binds the leader during import. A bacteria two-hybrid assay was here employed to determine if this could be an alternative way to investigate the binding of leader to the receptor. Leucine to alanine and arginine to glutamine mutations were made in the leader sequence from rat liver aldehyde dehydrogenase (pALDH). The leucine residues in the C-terminal of pALDH leader were found to be essential for TOM20 binding. The hydrophobic residues of another mitochondrial leader F1beta-ATPase that were important for Tom20 binding were found at the C-terminus of the leader. In contrast, it was the leucines in the N-terminus of the leader of ornithine transcarbamylase that were essential for binding. Modeling the peptides to the structure of Tom20 showed that the hydrophobic residues from the three proteins could all fit into the hydrophobic binding pocket. The mutants of pALDH that did not bind to Tom20 were still imported in vivo in transformed HeLa cells or in vitro into isolated mitochondria. In contrast, the mutant from pOTC was imported less well ( approximately 50%) while the mutant from F1beta-ATPase was not imported to any measurable extent. Binding to Tom20 might not be a prerequisite for import; however, it also is possible that import can occur even if binding to a receptor component is poor, so long as the leader binds tightly to another component of the translocator.


Subject(s)
Hydrophobic and Hydrophilic Interactions , Mitochondrial Proteins/metabolism , Protein Interaction Mapping/methods , Protein Sorting Signals/physiology , Protein Transport/physiology , Receptors, Cytoplasmic and Nuclear/metabolism , Aldehyde Dehydrogenase/chemistry , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase/metabolism , Amino Acid Sequence , Animals , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Membrane Transport Proteins , Microscopy, Confocal , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proton-Translocating ATPases/metabolism , Models, Molecular , Mutant Proteins/metabolism , Ornithine Carbamoyltransferase/metabolism , Protein Binding , Protein Sorting Signals/genetics , Proton-Translocating ATPases/metabolism , Rats , Receptors, Cell Surface , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques
18.
Biochemistry ; 45(31): 9445-53, 2006 Aug 08.
Article in English | MEDLINE | ID: mdl-16878979

ABSTRACT

Random mutagenesis followed by a filter-based screening assay has been used to identify a mutant of human class 1 aldehyde dehydrogenase (ALDH1) that was no longer inhibited by Mg(2+) ions but was activated in their presence. Several mutants possessed double, triple, and quadruple amino acid substitutions with a total of seven different residues being altered, but each had a common T244S change. This point mutation proved to be responsible for the Mg(2+) ion activation. An ALDH1 T244S mutant was recombinantly expressed and was used for mechanistic studies. Mg(2+) ions have been shown to increase the rate of deacylation. Consistent with the rate-limiting step for ALDH1 being changed from coenzyme dissociation to deacylation was finding that chloroacetaldehyde was oxidized more rapidly than acetaldehyde. Furthermore, Mg(2+) ions only in the presence of NAD(H) increased the rate of hydrolysis of p-nitrophenyl acetate showing that the metal only affects the binary complex. Though the rate-limiting step for the T244S mutant was different from that of the native enzyme, the catalytic efficiency of the mutant was just 20% that of the native enzyme. The basis for the change in the rate-limiting step appears to be related to NAD(+) binding. Using the structure of a sheep class 1 ALDH, it was possible to deduce that the interaction between the side chain of T244 and its neighboring residues with the nicotinamide ring of NAD(+) were an essential determinant in the catalytic action of ALDH1.


Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase/chemistry , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Magnesium/pharmacology , Aldehyde Dehydrogenase/genetics , Aldehyde Dehydrogenase 1 Family , Animals , Catalysis , Cations, Divalent/pharmacology , Computational Biology , Cytosol/enzymology , Enzyme Activation/genetics , Esterases/antagonists & inhibitors , Esterases/drug effects , Humans , Hydrolysis , Isoenzymes/genetics , Magnesium/chemistry , Models, Molecular , Mutagenesis , NAD/chemistry , Nitrophenols/chemistry , Point Mutation , Protein Conformation , Retinal Dehydrogenase , Sheep
19.
Protein Sci ; 15(6): 1387-96, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16731973

ABSTRACT

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step.


Subject(s)
Aldehyde Oxidoreductases/chemistry , Aldehyde Oxidoreductases/metabolism , Escherichia coli Proteins/chemistry , Amino Acid Sequence , Escherichia coli Proteins/metabolism , Esterases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Weight , NAD/metabolism , NADP/metabolism , Protein Conformation , Sequence Homology, Amino Acid , Structural Homology, Protein , Substrate Specificity
20.
Rejuvenation Res ; 9(2): 182-90, 2006.
Article in English | MEDLINE | ID: mdl-16706640

ABSTRACT

The human mitochondrion contains a small circular genome that codes for 13 proteins, 22 tRNAs, and 2 rRNAs. The proteins are all inner membrane bound components of complexes involved in the electron transport system and ATP formation. Mutations to any of the 13 proteins affect cellular behavior because energy production could be decreased. Investigators have attempted to find methods to correct these mutated proteins. One way is to express the mitochondrial gene in the nucleus (called allotopic expression). The newly synthesized protein would have to be imported into mitochondria and assembled into complexes. This paper reviews some of the successful attempts to achieve allotopic expression and discusses some issues that might affect the ability to have the proteins properly inserted into the inner membrane.


Subject(s)
DNA Damage/physiology , Mitochondrial Membranes/physiology , Mitochondrial Proteins/physiology , Animals , Humans , Hydrophobic and Hydrophilic Interactions , Mutation
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