Aldehyde dehydrogenases (ALDHs) are involved in the detoxification of aldehydes generated as byproducts of lipid peroxidation. In this work, it was determined that, among the three most studied human ALDH isoforms, ALDH2 showed the highest catalytic efficiency for oxidation of acrolein, 4-hydroxy-2-nonenal (4-HNE), and malondialdehyde. ALDH1A1 also exhibited significant activity with these substrates, whereas ALDH3A1 only showed activity with 4-HNE. ALDH2 was also the most sensitive isoform to irreversible inactivation by these compounds. Remarkably, ALDH3A1 was insensitive to these aldehydes even at concentrations as high as 20 mM. Formation of adducts of ALDH1A1 and ALDH2 with acrolein increased their K(d) values for NAD(+) by 2- and 3-fold, respectively. NADH exerted a higher protection than propionaldehyde to the inactivation by acrolein, and this protection was additive. These results suggested that both binding sites, those for aldehyde and NAD(+) in ALDH2, are targets for the inactivation by lipid peroxidation products. Thus, with the advantage of being relatively inactivation-insensitive, ALDH1A1 and ALDH3A1 may be actively participating in the detoxification of these aldehydes in the cells.

Many different diseases are associated with oxidative stress. One of the main consequences of oxidative stress at the cellular level is lipid peroxidation, from which toxic aldehydes may be generated. Below their toxicity thresholds, some aldehydes are involved in signaling processes, while others are intermediaries in the metabolism of lipids, amino acids, neurotransmitters, and carbohydrates. Some aldehydes ubiquitously distributed in the environment, such as acrolein or formaldehyde, are extremely toxic to the cell. On the other hand, aldehyde dehydrogenases (ALDHs) are able to detoxify a wide variety of aldehydes to their corresponding carboxylic acids, thus helping to protect from oxidative stress. ALDHs are located in different subcellular compartments such as cytosol, mitochondria, nucleus, and endoplasmic reticulum. The aim of this review is to analyze, and highlight, the role of different ALDH isoforms in the detoxification of aldehydes generated in processes that involve high levels of oxidative stress. The ALDH physiological relevance becomes evident by the observation that their expression and activity are enhanced in different pathologies that involve oxidative stress such as neurodegenerative disorders, cardiopathies, atherosclerosis, and cancer as well as inflammatory processes. Furthermore, ALDH mutations bring about several disorders in the cell. Thus, understanding the mechanisms by which these enzymes participate in diverse cellular processes may lead to better contend with the damage caused by toxic aldehydes in different pathologies by designing modulators and/or protocols to modify their activity or expression.

The modulation of aldehyde dehydrogenase (ALDH) activity has been suggested as a promising option for the prevention or treatment of many diseases. To date, only few activating compounds of ALDHs have been described. In this regard, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide has been used to protect the heart against ischemia/reperfusion damage. In the search for new modulating ALDH molecules, the binding capability of different compounds to the active site of human aldehyde dehydrogenase class 1A1 (ALDH1A1) was analyzed by molecular docking, and their ability to modulate the activity of the enzyme was tested. Surprisingly, tamoxifen, an estrogen receptor antagonist used for breast cancer treatment, increased the activity and decreased the Km for NAD(+) by about twofold in ALDH1A1. No drug effect on human ALDH2 or ALDH3A1 was attained, showing that tamoxifen was specific for ALDH1A1. Protection against thermal denaturation and competition with daidzin suggested that tamoxifen binds to the aldehyde site of ALDH1A1, resembling the interaction of N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide with ALDH2. Further kinetic analysis indicated that tamoxifen activation may be related to an increase in the Kd for NADH, favoring a more rapid release of the coenzyme, which is the rate-limiting step of the reaction for this isozyme. Therefore, tamoxifen might improve the antioxidant response, which is compromised in some diseases.

Mitochondrial aldehyde dehydrogenase (ALDH2) has been proposed as a key enzyme in cardioprotection during ischemia-reperfusion processes. This proposal led to the search for activators of ALDH2 with the aim to develop cardioprotective drugs. Alda-1 was the first activator of ALDH2 identified and its cardioprotective effect has been extensively proven in vivo; however, the mechanism of activation is not fully understood. A crystallographic study showed that Alda-1 binds to the entrance of the aldehyde-binding site; therefore, Alda-1 should in essence be an inhibitor. In the present study, kinetic experiments were performed to characterize the effect of Alda-1 on the properties of ALDH2 (kinetic parameters, determination of the rate-limiting step, reactivity of the catalytic cysteine) and on the kinetic mechanism (type of kinetics, sequence of substrates entering, and products release). The results showed that Alda-1 dramatically modifies the properties of ALDH2, the K for NAD decreased by 2.4-fold, and the catalytic efficiency increased 4.4-fold; however, the K for the aldehyde increased 8.6-fold, thus, diminishing the catalytic efficiency. The alterations in these parameters resulted in a complex behavior, where Alda-1 acts as inhibitor at low concentrations of aldehyde and as an activator at high concentrations. Additionally, the binding of Alda-1 to ALDH2 made the deacylation less limiting and diminished the pK of the catalytic cysteine. Finally, NADH inhibition patterns indicated that Alda-1 induced a change in the sequence of substrates entry and products release, in agreement with the proposal of both substrates entering ALDH2 by the NAD entrance site.

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 10 4 min −1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 10 3 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

Under physiological conditions, cells produce low basal levels of reactive oxygen species (ROS); however, in pathologic conditions ROS production increases dramatically, generating high concentrations of toxic unsaturated aldehydes. Aldehyde dehydrogenases (ALDHs) are responsible for detoxification of these aldehydes protecting the cell. Due to the physiological relevance of these enzymes, it is important to design strategies to modulate their activity. It was previously reported that omeprazole activation of ALDH1A1 protected Escherichia coli cells overexpressing this enzyme, from oxidative stress generated by H 2 O 2 . In this work, omeprazole cell protection potential was evaluated in eukaryotic cells. AS-30D cell or hepatocyte suspensions were subjected to a treatment with omeprazole and exposure to light (that is required to activate omeprazole in the active site of ALDH) and then exposed to H 2 O 2 . Cells showed viability similar to control cells, total activity of ALDH was preserved, while cell levels of lipid aldehydes and oxidative stress markers were maintained low. Cell protection by omeprazole was avoided by inhibition of ALDHs with disulfiram, revealing the key role of these enzymes in the protection. Additionally, omeprazole also preserved ALDH2 (mitochondrial isoform) activity, diminishing lipid aldehyde levels and oxidative stress in this organelle, protecting mitochondrial respiration and transmembrane potential formation capacity, from the stress generated by H 2 O 2 . These results highlight the important role of ALDHs as part of the antioxidant system of the cell, since if the activity of these enzymes decreases under stress conditions, the viability of the cell is compromised.

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Ethanol is one of the most efficient carbon sources for Euglena gracilis. Thus, an in-depth investigation of the distribution of ethanol metabolizing enzymes in this organism was conducted. Cellular fractionation indicated localization of the ethanol metabolizing enzymes in both cytosol and mitochondria. Isolated mitochondria were able to generate a transmembrane electrical gradient (Δψ) after the addition of ethanol. However, upon the addition of acetaldehyde no Δψ was formed. Furthermore, acetaldehyde collapsed Δψ generated by ethanol or malate but not by D-lactate. Pyrazole, a specific inhibitor of alcohol dehydrogenase (ADH), abolished the effect of acetaldehyde on Δψ, suggesting that the mitochondrial ADH, by actively consuming NADH to reduce acetaldehyde to ethanol, was able to collapse Δψ. When mitochondria were fractionated, 27% and 60% of ADH and aldehyde dehydrogenase (ALDH) activities were found in the inner membrane fraction. ADH activity showed two kinetic components, suggesting the presence of two isozymes in the membrane fraction, while ALDH kinetics was monotonic. The ADH Km values were 0.64-6.5 mM for ethanol, and 0.16-0.88 mM for NAD+, while the ALDH Km values were 1.7-5.3 μM for acetaldehyde and 33-47 μM for NAD+. These novel enzymes were also able to use aliphatic substrates of different chain length and could be involved in the metabolism of fatty alcohol and aldehydes released from wax esters stored by this microorganism.