Henry Weiner

Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion.

BACKGROUND The single genetic factor most strongly correlated with reduced alcohol consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The E487K variant of ALDH2 is found in approximately 50% of the Asian population, and is associated with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. The structure of ALDH2 is important for the elucidation of its catalytic mechanism, to gain a clear understanding of the contribution of ALDH2 to the genetic component of alcoholism and for the development of specific ALDH2 inhibitors as potential drugs for use in the treatment of alcoholism. RESULTS The X-ray structure of bovine ALDH2 has been solved to 2.65 A in its free form and to 2.75 A in a complex with NAD+. The enzyme structure contains three domains; two dinucleotide-binding domains and a small three-stranded beta-sheet domain, which is involved in subunit interactions in this tetrameric enzyme. The E487K mutation occurs in this small oligomerization domain and is located at a key interface between subunits immediately below the active site of another monomer. The active site of ALDH2 is divided into two halves by the nicotinamide ring of NAD+. Adjacent to the A-side (Pro-R) of the nicotinamide ring is a cluster of three cysteines (Cys301, Cys302 and Cys303) and adjacent to the B-side (Pro-S) are Thr244, Glu268, Glu476 and an ordered water molecule bound to Thr244 and Glu476. CONCLUSIONS Although there is a recognizable Rossmann-type fold, the coenzyme-binding region of ALDH2 binds NAD+ in a manner not seen in other NAD+-binding enzymes. The positions of the residues near the nicotinamide ring of NAD+ suggest a chemical mechanism whereby Glu268 functions as a general base through a bound water molecule. The sidechain amide nitrogen of Asn169 and the peptide nitrogen of Cys302 are in position to stabilize the oxyanion present in the tetrahedral transition state prior to hydride transfer. The functional importance of residue Glu487 now appears to be due to indirect interactions of this residue with the substrate-binding site via Arg264 and Arg475.

Mitochondrial Aldehyde Dehydrogenase Activity Is Required for Male Fertility in Maize

Some plant cytoplasms express novel mitochondrial genes that cause male sterility. Nuclear genes that disrupt the accumulation of the corresponding mitochondrial gene products can restore fertility to such plants. The Texas (T) cytoplasm mitochondrial genome of maize expresses a novel protein, URF13, which is necessary for T cytoplasm–induced male sterility. Working in concert, functional alleles of two nuclear genes, rf1 and rf2, can restore fertility to T cytoplasm plants. Rf1 alleles, but not Rf2 alleles, reduce the accumulation of URF13. Hence, Rf2 differs from typical nuclear restorers in that it does not alter the accumulation of the mitochondrial protein necessary for T cytoplasm–induced male sterility. This study established that the rf2 gene encodes a soluble protein that accumulates in the mitochondrial matrix. Three independent lines of evidence establish that the RF2 protein is an aldehyde dehydrogenase (ALDH). The finding that T cytoplasm plants that are homozygous for the rf2-R213 allele are male sterile but accumulate normal amounts of RF2 protein that lacks normal mitochondrial (mt) ALDH activity provides strong evidence that rf2-encoded mtALDH activity is required to restore male fertility to T cytoplasm maize. Detailed genetic analyses have established that the rf2 gene also is required for anther development in normal cytoplasm maize. Hence, it appears that the rf2 gene was recruited recently to function as a nuclear restorer. ALDHs typically have very broad substrate specificities. Indeed, the RF2 protein is capable of oxidizing at least three aldehydes. Hence, the specific metabolic pathway(s) within which the rf2-encoded mtALDH acts remains to be discovered.

Role of reduced lipoic acid in the redox regulation of mitochondrial aldehyde dehydrogenase (ALDH-2) activity : Implications for mitochondrial oxidative stress and nitrate tolerance

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 μg/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.

In vivo acetaldehyde in the brain of the rat treated with ethanol

Abstract Ethanol, 4.5 g/kg, was administered intragastrically to rats to determine if acetaldehyde could be detected in brain interstitial fluid. Samples from both blood and brain were collected at half-hour intervals. Brain interstitial fluid samples were collected from both the caudate nucleus and the thalamushypothalamus region using the push-pull perfusion technique. The ethanol and acetaldehyde concentrations in these samples were determined by a head space gas Chromatographie technique. Blood ethanol levels typically ranged from 200 to 400 mg/100ml, while acetaldehyde levels ranged from 15 to 40 μM in blood and 5 to 20 μM in brain fluid. When disulfiram was given to the rats 20 hr prior to ethanol administration, blood acetaldehyde increased to 70–280 μM and brain interstitial fluid acetaldehyde increased to between 25 and 120 μM. Whole brain acetaldehyde levels were also measured after an ethanol dose was given. No acetaldehyde could be detected in whole brain unless the animal had first been treated with disulfiram. These data demonstrate that acetaldehyde does enter the brain, coming into direct contact with the brain cells bathed in the interstitial fluid. The acetaldehyde concentration in the interstitial fluid is higher than that in the brain cells, probably due to its rapid oxidation in the cells catalyzed by aldehyde dehydrogenase.

Enzymology and subcellular localization of aldehyde oxidation in rat liver. Oxidation of 3,4-dihydroxyphenylacetaldehyde derived from dopamine to 3,4-dihydroxyphenylacetic acid.

Abstract In the presence of ethanol, the metabolism of dopamine in rat liver slices is altered such that the major product is 3,4-dihydroxyphenylethanol, not 3,4-dihydroxyphenylacetic acid (DOPAC). It has been proposed that this metabolic alteration is due to the inhibition of the oxidation of 3,4-dihydroxyphenylacetaldehyde (DOPAL) by acetaldehyde, the first metabolite of ethanol. The oxidation of DOPAL in rat liver slices, however, is not inhibited dramatically by relatively high concentrations of acetaldehyde [A. W. Tank and H. Weiner, Biochem. Pharmac. 28 , 3139 (1979)]. Thus, it is possible that acetaldehyde and DOPAL are oxidized by different isozymes of aldehyde dehydrogenase (ALDH) present in different subcellular compartments. Acetaldehyde is oxidized by isozymes of ALDH that are found in the matrix space of the mitochondria of rat liver. The subcellular site of the oxidation of most other aldehydes is not known. Mitochondrial, microsomal and cytosol fractions of the rat liver were isolated by differential centrifugation, and the isozymes of ALDH present in the cytosol and mitochondrial fractions were separated by column isoelectric focusing. Five isozymes of ALDH were isolated from the cytosol, and three isozymes were isolated from the mitochondria. The K m values for acetaldehyde, p -nitrobenzaldehyde and DOPAL for each of the isolated isozymes were determined and were found to range from approximately 1 μM to 1 mM. Each subcellular fraction was incubated with [ethylamine-2- 14 C]dopamine to determine its ability to oxidize DOPAL. Partially purified monoamine oxidase was used to generate DOPAL for those incubations which did not contain mitochondria. Intact mitochondria were capable of oxidizing virtually all the DOPAL to DOPAC in the presence or absence of added pyridine nucleotide coenzymes. Cytosol and microsomal fractions were capable of oxidizing the aldehyde, but not to the same extent as the intact mitochondria. ALDH activity present in the mitochondrial matrix space was inhibited by the addition of rotenone. This treatment inhibited formation of DOPAC by 80 per cent in isolated intact mitochondria in the absence of added pyridine nucleotides. Inclusion of rotenone caused the inhibition of DOPAC formation by ca. 50 per cent when intact mitochondria, microsomes and cytosol were incubated together with dopamine. These results suggest that an isozyme of ALDH present in the mitochondrial matrix space is primarily responsible for the oxidation of DOPAL in rat liver, though nonmitochondrial enzymes can contribute to the oxidation.

Involvement of Glutamate 399 and Lysine 192 in the Mechanism of Human Liver Mitochondrial Aldehyde Dehydrogenase

Mutation to the conserved Glu399 or Lys192 caused the rate-limiting step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurley, T. D., and Weiner, H. (1997) J. Biol. Chem.272, 18817–18822). Here we further investigated the role of these two NAD+-ribose-binding residues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase activity when compared with the native enzyme. No pre-steady state burst of NADH formation was found with the E399Q/K and K192Q enzymes when propionaldehyde was used as the substrate; furthermore, each mutant oxidized chloroacetaldehyde slower than propionaldehyde, and a primary isotope effect was observed for each mutant when [2H]acetaldehyde was used as a substrate. However, no isotope effect was observed for each mutant when α-[2H]benzaldehyde was the substrate. A pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants. The rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates. The K192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.

The mutation in the mitochondrial aldehyde dehydrogenase (ALDH2) gene responsible for alcohol-induced flushing increases turnover of the enzyme tetramers in a dominant fashion.

Deficiency in mitochondrial aldehyde dehydrogenase (ALDH2), a tetrameric enzyme, results from inheriting one or two ALDH2*2 alleles. This allele encodes a protein subunit with a lysine for glutamate substitution at position 487 and is dominant over the wild-type allele, ALDH2*1. The ALDH2*2-encoded subunit (ALDH2K) reduces the activity of ALDH2 enzyme in cell lines expressing the wild-type subunit (ALDH2E). In addition to this effect on the enzyme activity, we now report that ALDH2*2 heterozygotes had lower levels of ALDH2 immunoreactive protein in autopsy liver samples. The half-lives of ALDH2 protein in HeLa cell lines expressing ALDH2*1, ALDH2*2, or both were determined by the rate of loss of immunoreactive protein after inhibition of protein synthesis with puromycin and by pulse-chase experiments. By either measure, ALDH2E enzyme was very stable, with a half-life of at least 22 h. ALDH2K enzyme had an enzyme half-life of only 14 h. In cells expressing both subunits, most of the subunits assemble as heterotetramers, and these enzymes had a half-life of 13 h. Thus, the effect of ALDH2K on enzyme turnover is dominant. These studies indicate that the ALDH2*2 allele exerts its dominant effect both by interfering with the catalytic activity of the enzyme and by increasing its turnover. This represents the first example of a dominantly acting allele with this effect on a mitochondrial enzymes turnover.

Structural and functional consequences of coenzyme binding to the inactive asian variant of mitochondrial aldehyde dehydrogenase: roles of residues 475 and 487.

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 kcat for these processes and increases the Km 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 2.5Å. 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 coenzymebinding 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.

Heme Oxygenase-1. A Novel Key Player in the Development of Tolerance in Response to Organic Nitrates

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 μg/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.

In Vivo Mitochondrial Import A COMPARISON OF LEADER SEQUENCE CHARGE AND STRUCTURAL RELATIONSHIPS WITH THE IN VITRO MODEL RESULTING IN EVIDENCE FOR CO-TRANSLATIONAL IMPORT

The positive charges and structural properties of the mitochondrial leader sequence of aldehyde dehydrogenase have been extensively studied in vitro. The results of these studies showed that increasing the helicity of this leader would compensate for reduced import from positive charge substitutions of arginine with glutamine or the insertion of negative charged residues made in the native leader. In this in vivo study, utilizing the green fluorescent protein (GFP) as a passenger protein, import results showed the opposite effect with respect to helicity, but the results from mutations made within the native leader sequence were consistent between the in vitro and in vivo experiments. Leader mutations that reduced the efficiency of import resulted in a cytosolic accumulation of a truncated GFP chimera that was fluorescent but devoid of a mitochondrial leader. The native leader efficiently imported before GFP could achieve a stable, import-incompetent structure, suggesting that import was coupled with translation. As a test for a co-translational mechanism, a chimera of GFP that contained the native leader of aldehyde dehydrogenase attached at the N terminus and a C-terminal endoplasmic reticulum targeting signal attached to the C terminus of GFP was constructed. This chimera was localized exclusively to mitochondria. The import result with the dual signal chimera provides support for a co-translational mitochondrial import pathway.