Coenzyme specificity in aldehyde dehydrogenase

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2'-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2'- and 3'-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2'-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2'-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2'-phosphate of NADP.     

Coenzyme specificity in aldehyde dehydrogenase

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2′-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2′- and 3′-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2′-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2′-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2′-phosphate of NADP.     

Aldehyde dehydrogenase. Maintaining critical active site geometry at motif 8 in the class 3 enzyme.

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.     

Apo and holo crystal structures of an NADP-dependent aldehyde dehydrogenase from Streptococcus mutans.

The aldehyde dehydrogenases (ALDHs) are a superfamily of multimeric enzymes which catalyse the oxidation of a broad range of aldehydes into their corresponding carboxylic acids with the reduction of their cofactor, NAD or NADP, into NADH or NADPH. At present, the only known structures concern NAD-dependent ALDHs. Three structures are available in the Protein Data Bank: two are tetrameric and the other is a dimer. We solved by molecular replacement the first structure of an NADP-dependent ALDH isolated from Streptococcus mutans, in its apo form and holo form in complex with NADP, at 1.8 and 2.6 A resolution, respectively. Although the protein sequence shares only approximately 30 % identity with the other solved tetrameric ALDHs, the structures are very similar. However, a large local conformational change in the region surrounding the 2' phosphate group of the adenosine moiety is observed when the enzyme binds NADP, in contrast to the NAD-dependent ALDHs.Structure and sequence analyses reveal several properties. A small number of residues seem to determine the oligomeric state. Likewise, the nature (charge and volume) of the residue at position 180 (Thr in ALDH from S. mutans) determines the cofactor specificity in comparison with the structures of NAD-dependent ALDHs. The presence of a hydrogen bond network around the cofactor not only allows it to bind to the enzyme but also directs the side-chains in a correct orientation for the catalytic reaction to take place. Moreover, a specific part of this network appears to be important in substrate binding. Since the enzyme oxidises the same substrate, glyceraldehyde-3-phosphate (G3P), as NAD-dependent phosphorylating glyceraldehyde-3-phosphate dehydrogenases (GAPDH), the active site of GAPDH was compared with that of the S. mutans ALDH. It was found that Arg103, Arg283 and Asp440 might be key residues for substrate binding.     

Engineering the nucleotide coenzyme specificity and sulfhydryl redox sensitivity of two stress-responsive aldehyde dehydrogenase isoenzymes of Arabidopsis thaliana

Lipid peroxidation is one of the consequences of environmental stress in plants and leads to the accumulation of highly toxic, reactive aldehydes. One of the processes to detoxify these aldehydes is their oxidation into carboxylic acids catalyzed by NAD(P)+-dependent ALDHs (aldehyde dehydrogenases). We investigated kinetic parameters of two Arabidopsis thaliana family 3 ALDHs, the cytosolic ALDH3H1 and the chloroplastic isoform ALDH3I1. Both enzymes had similar substrate specificity and oxidized saturated aliphatic aldehydes. Catalytic efficiencies improved with the increase of carbon chain length. Both enzymes were also able to oxidize α,β-unsaturated aldehydes, but not aromatic aldehydes. Activity of ALDH3H1 was NAD+-dependent, whereas ALDH3I1 was able to use NAD+ and NADP+. An unusual isoleucine residue within the coenzyme-binding cleft was responsible for the NAD+-dependence of ALDH3H1. Engineering the coenzyme-binding environment of ALDH3I1 elucidated the influence of the surrounding amino acids. Enzyme activities of both ALDHs were redox-sensitive. Inhibition was correlated with oxidation of both catalytic and non-catalytic cysteine residues in addition to homodimer formation. Dimerization and inactivation could be reversed by reducing agents. Mutant analysis showed that cysteine residues mediating homodimerization are located in the N-terminal region. Modelling of the protein structures revealed that the redox-sensitive cysteine residues are located at the surfaces of the subunits.     

Relationships within the aldehyde dehydrogenase extended family

One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.     

Adenine Binding Mode Is a Key Factor in Triggering the Early Release of NADH in Coenzyme A-dependent Methylmalonate Semialdehyde Dehydrogenase

Structural dynamics associated with cofactor binding have been shown to play key roles in the catalytic mechanism of hydrolytic NAD(P)-dependent aldehyde dehydrogenases (ALDH). By contrast, no information is available for their CoA-dependent counterparts. We present here the first crystal structure of a CoA-dependent ALDH. The structure of the methylmalonate semialdehyde dehydrogenase (MSDH) from Bacillus subtilis in binary complex with NAD(+) shows that, in contrast to what is observed for hydrolytic ALDHs, the nicotinamide ring is well defined in the electron density due to direct and H(2)O-mediated hydrogen bonds with the carboxamide. The structure also reveals that a conformational isomerization of the NMNH is possible in MSDH, as shown for hydrolytic ALDHs. Finally, the adenine ring is substantially more solvent-exposed, a result that could be explained by the presence of a Val residue at position 229 in helix α(F) that reduces the depth of the binding pocket and the absence of Gly-225 at the N-terminal end of helix α(F). Substitution of glycine for Val-229 and/or insertion of a glycine residue at position 225 resulted in a significant decrease of the rate constant associated with the dissociation of NADH from the NADH/thioacylenzyme complex, thus demonstrating that the weaker stabilization of the adenine ring is a key factor in triggering the early NADH release in the MSDH-catalyzed reaction. This study provides for the first time structural insights into the mechanism whereby the cofactor binding mode is responsible at least in part for the different kinetic behaviors of the hydrolytic and CoA-dependent ALDHs.     

The ALDH gene superfamily of Arabidopsis

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily of NAD(P)(+)-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. The Arabidopsis genome contains 14 unique ALDH sequences encoding members of nine ALDH families, including eight known families and one novel family (ALDH22) that is currently known only in plants. Here, we identify members of the ALDH gene superfamily in Arabidopsis; provide a revised, unified nomenclature for these ALDH genes; analyze the molecular relationship among Arabidopsis ALDH genes and compare them to ALDH genes from other species, including prokaryotes and mammals; and describe the role of ALDHs in cytoplasmic male sterility, plant defense and abiotic stress tolerance.     

The soybean aldehyde dehydrogenase (ALDH) protein superfamily

Aldehyde dehydrogenases (ALDHs) are members of NAD(P)(+)-dependent protein superfamily that catalyze the oxidation of a wide range of endogenous and exogenous highly reactive aliphatic and aromatic aldehyde molecules to their corresponding non toxic carboxylic acids. Research evidence has shown that ALDHs represent a promising class of genes to improve growth development, seed storage and environmental stress adaptation in higher plants. The recently completed genome sequences of several plant species have resulted in the identification of a large number of ALDH genes, most of which still need to be functionally characterized. In this paper, we identify members of the ALDH gene superfamily in soybean genome, and provide a unified nomenclature for the entire soybean ALDH gene families. The soybean genome contains 18 unique ALDH sequences encoding members of five ALDH families involved in a wide range of metabolic and molecular detoxification pathways. In addition, we describe the biochemical requirements and cellular metabolic pathways of selected members of ALDHs in soybean responses to environmental stress conditions.     

Group X aldehyde dehydrogenases of Pseudomonas aeruginosa PAO1 degrade hydrazones

Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD(+)- or NADP(+)-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological roles of the ALDH family remain unknown. The PAO1 strain upregulated HDH in the presence of the hydrazone adipic acid bis(ethylidene hydrazide) (AEH). Gene disruption of the HDH-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double gene disruption of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of group X ALDHs from Escherichia coli, Paracoccus denitrificans, and Ochrobactrum anthropi also acted as HDHs in that they produced HDH activity in the cells and degraded hydrazones. These findings indicated the physiological roles of group X ALDHs in bacteria and showed that they comprise a distinct ALDH subfamily.     

Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase.

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.     

Discovery of a novel class of covalent inhibitor for aldehyde dehydrogenases

Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated β-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.     

Human aldehyde dehydrogenase-catalyzed oxidation of ethylene glycol ether aldehydes

Ethylene glycol ethers (EGEs) are primary alcohols commonly used as solvents in numerous household and industrial products. Exposure to EGEs has been correlated with delayed encephalopathy, metabolic acidosis, sub-fertility and spermatotoxicity in humans. In addition, they also cause teratogenesis, carcinogenesis, hemolysis, etc., in various animal models. Metabolism EGEs parallels ethanol metabolism, i.e., EGEs are first converted to 2-alkoxy acetaldehydes (EGE aldehydes) by alcohol dehydrogenases, and then to alkoxyacetic acids by aldehyde dehydrogenases (ALDHs). The acid metabolite of EGEs is considered responsible for toxicities associated with EGEs. The role of human ALDHs in EGE metabolism is not clear; accordingly, we have investigated the ability of five different human ALDHs (ALDH1A1, ALDH2, ALDH3A1, ALDH5A1 and ALDH9A1) to catalyze the oxidation of various EGE aldehydes. The EGE aldehydes used in this study were synthesized via Swern oxidation. All of the human ALDHs were purified from human cDNA clones over-expressing these enzymes in E. coli. The ALDHs tested, so far, differentially catalyze the oxidation of EGE aldehydes to their corresponding acids (K(m) values range from approximately 10 microM to approximately 20.0mM). As judged by V(max)/K(m) ratios, short-chain alkyl-group containing EGE aldehydes are oxidized to their acids more efficiently by ALDH2, whereas aryl- and long-chain alkyl-group containing EGE aldehydes are oxidized to their acid more efficiently by ALDH3A1. Given the product of ALDH-catalyzed reaction is toxic, this process should be considered as a bio-activation (toxification) process.     

Post-translational modifications of mitochondrial aldehyde dehydrogenase and biomedical implications

Aldehyde dehydrogenases (ALDHs) represent large family members of NAD(P)+-dependent dehydrogenases responsible for the irreversible metabolism of many endogenous and exogenous aldehydes to the corresponding acids. Among 19 ALDH isozymes, mitochondrial ALDH2 is a low Km enzyme responsible for the metabolism of acetaldehyde and lipid peroxides such as malondialdehyde and 4-hydroxynonenal, both of which are highly reactive and toxic. Consequently, inhibition of ALDH2 would lead to elevated levels of acetaldehyde and other reactive lipid peroxides following ethanol intake and/or exposure to toxic chemicals. In addition, many East Asian people with a dominant negative mutation in ALDH2 gene possess a decreased ALDH2 activity with increased risks for various types of cancer, myocardial infarct, alcoholic liver disease, and other pathological conditions. The aim of this review is to briefly describe the multiple post-translational modifications of mitochondrial ALDH2, as an example, after exposure to toxic chemicals or under different disease states and their pathophysiological roles in promoting alcohol/drug-mediated tissue damage. We also briefly mention exciting preclinical translational research opportunities to identify small molecule activators of ALDH2 and its isozymes as potentially therapeutic/preventive agents against various disease states where the expression or activity of ALDH enzymes is altered or inactivated.     

Aldehyde dehydrogenases and cell proliferation

Aldehyde dehydrogenases (ALDHs) oxidize aldehydes to the corresponding carboxylic acids using either NAD or NADP as a coenzyme. Aldehydes are highly reactive aliphatic or aromatic molecules that play an important role in numerous physiological, pathological, and pharmacological processes. ALDHs have been discovered in practically all organisms and there are multiple isoforms, with multiple subcellular localizations. More than 160 ALDH cDNAs or genes have been isolated and sequenced to date from various sources, including bacteria, yeast, fungi, plants, and animals. The eukaryote ALDH genes can be subdivided into several families; the human genome contains 19 known ALDH genes, as well as many pseudogenes. Noteworthy is the fact that elevated activity of various ALDHs, namely ALDH1A2, ALDH1A3, ALDH1A7, ALDH2*2, ALDH3A1, ALDH4A1, ALDH5A1, ALDH6, and ALDH9A1, has been observed in normal and cancer stem cells. Consequently, ALDHs not only may be considered markers of these cells, but also may well play a functional role in terms of self-protection, differentiation, and/or expansion of stem cell populations. The ALDH3 family includes enzymes able to oxidize medium-chain aliphatic and aromatic aldehydes, such as peroxidic and fatty aldehydes. Moreover, these enzymes also have noncatalytic functions, including antioxidant functions and some structural roles. The gene of the cytosolic form, ALDH3A1, is localized on chromosome 17 in human beings and on the 11th and 10th chromosome in the mouse and rat, respectively. ALDH3A1 belongs to the phase II group of drug-metabolizing enzymes and is highly expressed in the stomach, lung, keratinocytes, and cornea, but poorly, if at all, in normal liver. Cytosolic ALDH3 is induced by polycyclic aromatic hydrocarbons or chlorinated compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, in rat liver cells and increases during carcinogenesis. It has been observed that this increased activity is directly correlated with the degree of deviation in hepatoma and lung cancer cell lines, as is the case in chemically induced hepatoma in rats. High ALDH3A1 expression and activity have been correlated with cell proliferation, resistance against aldehydes derived from lipid peroxidation, and resistance against drug toxicity, such as oxazaphosphorines. Indeed, cells with a high ALDH3A1 content are more resistant to the cytostatic and cytotoxic effects of lipidic aldehydes than are those with a low content. A reduction in cell proliferation can be observed when the enzyme is directly inhibited by the administration of synthetic specific inhibitors, antisense oligonucleotides, or siRNA or indirectly inhibited by the induction of peroxisome proliferator-activated receptor γ (PPARγ) with polyunsaturated fatty acids or PPARγ transfection. Conversely, cell proliferation is stimulated by the activation of ALDH3A1, whether by inhibiting PPARγ with a specific antagonist, antisense oligonucleotides, siRNA, or a medical device (i.e., composite polypropylene prosthesis for hernia repair) used to induce cell proliferation. To date, the mechanisms underlying the effects of ALDHs on cell proliferation are not yet fully clear. A likely hypothesis is that the regulatory effect is mediated by the catabolism of some endogenous substrates deriving from normal cell metabolism, such as 4-hydroxynonenal, which have the capacity to either stimulate or inhibit the expression of genes involved in regulating proliferation.     

An aspartate residue in yeast alcohol dehydrogenase I determines the specificity for coenzyme

In the three-dimensional structures of enzymes that bind NAD or FAD, there is an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme. The size and charge of the carboxylate might repel the binding of the 2'-phosphate group of NADP and explain the specificity for NAD. In the NAD-dependent alcohol dehydrogenases, Asp-223 (horse liver alcohol dehydrogenase sequence) appears to have this role. The homologous residue in yeast alcohol dehydrogenase I (residue 201 in the protein sequence) was substituted with Gly, and the D223G enzyme was expressed in yeast, purified, and characterized. The wild-type enzyme is specific for NAD. In contrast, the D223G enzyme bound and reduced NAD+ and NADP+ equally well, but, relative to wild-type enzyme, the dissociation constant for NAD+ was increased 17-fold, and the reactivity (V/K) on ethanol was decreased to 1%. Even though catalytic efficiency was reduced, yeast expressing the altered or wild-type enzyme grew at comparable rates, suggesting that equilibration of NAD and NADP pools is not lethal. Asp-223 participates in binding NAD and in excluding NADP, but it is not the only residue important for determining specificity for coenzyme.     

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 μM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10–25 μM on rat cultured hepatoma cells. Four hours after treatment with 25 μM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.     

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.     

Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily

The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+). The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety. The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism. However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile. As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile. The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.