Showing papers on "Aldehyde dehydrogenase published in 1998"

Human aldehyde dehydrogenase gene family

Abstract: Twelve aldehyde dehydrogenase (ALDH) genes have been identified in humans. These genes, located on different chromosomes, encode a group of enzymes which oxidizes varieties of aliphatic and aromatic aldehydes. Metabolic disorders and clinical problems associated with mutations of ALDH1, ALDH2, ALDH4, ALDH10 and succinic semialdehyde (SSDH) genes have been emerged. Comparison of the human ALDHs indicates a wide range of divergency (> 80−< 15 % identity at the protein sequence level) among them. However, several protein regions, some of which are implicated in functional activities, are conserved in the family members. The phylogenic tree constructed of 56 ALDH sequences of humans, animals, fungi, protozoa and eubacteria, suggests that the present-day human ALDH genes were derived from four ancestral genes that existed prior to the divergence of Eubacteria and Eukaryotes. The neighbor-joining tree derived from 12 human ALDHs and antiquitin indicates that diversification within the ALDH1/2/5/6 gene cluster occurred during the Neoproterozoic period (about 800 million years ago). Duplication in the ALDH 3/10/7/8 gene cluster occurred in Phanerozoic period (about 300 million years ago). Separations of ALDH3/ALDH10 and that of ALDH7/ALDH8 had occurred during the period of appearance and radiation of mammalian species.

Structure of betaine aldehyde dehydrogenase at 2.1 A resolution.

Abstract: The three-dimensional structure of betaine aldehyde dehydrogenase, the most abundant aldehyde dehydrogenase (ALDH) of cod liver, has been determined at 2.1 A resolution by the X-ray crystallographic method of molecular replacement. This enzyme represents a novel structure of the highly multiple ALDH, with at least 12 distinct classes in humans. This betaine ALDH of class 9 is different from the two recently determined ALDH structures (classes 2 and 3). Like these, the betaine ALDH structure has three domains, one coenzyme binding domain, one catalytic domain, and one oligomerization domain. Crystals grown in the presence or absence of NAD+ have very similar structures and no significant conformational change occurs upon coenzyme binding. This is probably due to the tight interactions between domains within the subunit and between subunits in the tetramer. The oligomerization domains link the catalytic domains together into two 20-stranded pleated sheet structures. The overall structure is similar to that of the tetrameric bovine class 2 and dimeric rat class 3 ALDH, but the coenzyme binding with the nicotinamide in anti conformation, resembles that of class 2 rather than of class 3.

Molecular Cloning, Characterization, and Potential Roles of Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Ethanol Metabolism in Saccharomyces cerevisiae

Abstract: Yeast aldehyde dehydrogenase (ALDH) has been studied for many years. In the 1950s it was found that a K+ ion-activated enzyme existed, and many kinetic properties of the enzyme were studied by different investigators (2, 3, 21). Most of the interest was focused on understanding the metabolism of acetaldehyde derived from the oxidation of ethanol during the aerobic growth of the organism. More recently, a different form of ALDH was identified which primarily oxidized long-chain aldehydes and was not involved in acetaldehyde oxidation (1, 25). Our laboratory reported on the cloning and sequencing of a yeast ALDH (19) and suggested it was mitochondrial in origin. When we started to reinvestigate the project, we came to realize that the DNA we sequenced could not have been totally correct. Hence, we rescreened our libraries and found two new DNAs which would code for different enzyme forms. The complete Saccharomyces cerevisiae genome has recently been published (7), and it verified that our original clone was not correct but that the two new genes we had found were present. Here we report data on the cloning, expression, purification, and characterization of these two enzyme forms. Neither is the commercially available K+-activated enzyme. During growth on ethanol it is necessary for S. cerevisiae to convert two carbons from ethanol to acetate so they can be used by the glyoxylate system to incorporate the carbons into usable metabolites. The total genomic sequence of S. cerevisiae S288C revealed that seven genes could code for proteins which appear to be members of the ALDH family. The recombinantly expressed enzymes encoded by the two genes we cloned appeared to have properties similar to enzymes found by other investigators. One enzyme was shown to be localized to the mitochondria, with properties somewhat similar, but not identical, to those reported by Black in 1951 (2). This enzyme was also shown to exhibit properties different from those of the commercially available enzyme. The second enzyme was localized to the cytosol, and had properties very similar to those of the enzyme described by Seegmiller (20) and Dickinson (4). The data also suggest that the acetaldehyde produced during ethanol oxidation could be converted into acetate in the cytosol as well as in the mitochondria. The contribution of the enzymes may be influenced by the NADP/NAD ratio in the cells. Investigators have suggested that mitochondrial ALDH was involved in the oxidative metabolism of ethanol while cytosolic ALDH was responsible for the oxidation of acetaldehyde produced during fermentation (15, 18), although the bulk of acetaldehyde is reduced to ethanol. We have used the nomenclature previously adopted (31) to identify the yeast enzymes described in this study based on their amino acid sequence homologies with and enzymatic similarities to the mammalian enzymes. We have disrupted the cytosolic ALDH gene (designated ALD1) and two mitochondrial ALDH genes (designated ALD2 and ALD5) in the S. cerevisiae genome and found that ALDH1 and ALDH2 were indeed important for the metabolism of ethanol. Both ALDHs might be involved in the oxidation of acetaldehyde formed during fermentation. The physiological role of ALDH5 in acetaldehyde metabolism was not completely elucidated in this study.

Characteristics of aldehyde dehydrogenases of certain aerobic bacteria representing human colonic flora.

Abstract: We have proposed the existence of a bacteriocolonic pathway for ethanol oxidation resulting in high intracolonic levels of toxic and carcinogenic acetaldehyde. This study was aimed at determining the ability of the aldehyde dehydrogenases (ALDH) of aerobic bacteria representing human colonic flora to metabolize intracolonically derived acetaldehyde. The apparent Michaelis constant ( K m) values for acetaldehyde were determined in crude extracts of five aerobic bacterial strains, alcohol dehydrogenase (ADH) and ALDH activities of these bacteria at conditions prevailing in the human large intestine after moderate drinking were then compared. The effect of cyanamide, a potent inhibitor of mammalian ALDH, on bacterial ALDH activity was also studied. The apparent K m for acetaldehyde varied from 6.8 (NADP+ -linked ALDH of Escherichia coli IH 13369) to 205 μM (NAD+ -linked ALDH of Pseudomonas aeruginosa IH 35342), and maximal velocity varied from 6 nmol/min/mg (NAD+ -linked ALDH of Klebsiella pneumoniae IH 35385) to 39 nmol/min/mg (NAD+ -linked ALDH of Pseudomonas aeruginosa IH 35342). At pH 7.4, and at ethanol and acetaldehyde concentrations that may be prevalent in the human colon after moderate drinking, ADH activity in four out of five bacterial strains were 10–50 times higher than their ALDH activity. Cyanamide inhibited only NAD+ -linked ALDH activity of Pseudomonas aeruginosa IH 35342 at concentrations starting from 0.1 mM. We conclude that ALDHs of the colonic aerobic bacteria are able to metabolize endogenic acetaldehyde. However, the ability of ALDHs to metabolize intracolonic acetaldehyde levels associated with alcohol drinking is rather low. Large differences between ADH and ALDH activities of the bacteria found in this study may contribute to the accumulation of acetaldehyde in the large intestine after moderate drinking. ALDH activities of colonic bacteria were poorly inhibited by cyanamide. This study supports the crucial role of intestinal bacteria in the accumulation of intracolonic acetaldehyde after drinking alcohol. Individual variations in human colonic flora may contribute to the risk of alcohol-related gastrointestinal morbidity.

Human ocular aldehyde dehydrogenase isozymes: distribution and properties as major soluble proteins in cornea and lens.

Abstract: Human aldehyde dehydrogenase isozymes (ALDHs; EC 1.2.1.3) exhibit very high levels of activity in anterior eye tissues. Human corneal ALDH1 and ALDH3 isozymes are present as major soluble proteins (3% and 5%, respectively, of corneal soluble protein) and may play major roles in protecting the cornea against ultraviolet radiation (UVR)-induced tissue damage, as well as contributing directly to ultraviolet B (UV-B) photoreception. The human lens exhibits high levels of ALDH1 activity (1-2% of lens-soluble protein) and lower levels of ALDH3 activity. Kinetic analyses support a role for these enzymes in the metabolism of peroxidic aldehydes, which have been reported in ocular tissues.

Purification and Characterization of the Coniferyl Aldehyde Dehydrogenase from Pseudomonas sp. Strain HR199 and Molecular Characterization of the Gene

Abstract: The coniferyl aldehyde dehydrogenase (CALDH) of Pseudomonas sp. strain HR199 (DSM7063), which catalyzes the NAD+-dependent oxidation of coniferyl aldehyde to ferulic acid and which is induced during growth with eugenol as the carbon source, was purified and characterized. The native protein exhibited an apparent molecular mass of 86,000 +/- 5,000 Da, and the subunit mass was 49.5 +/- 2.5 kDa, indicating an alpha2 structure of the native enzyme. The optimal oxidation of coniferyl aldehyde to ferulic acid was obtained at a pH of 8.8 and a temperature of 26 degreesC. The Km values for coniferyl aldehyde and NAD+ were about 7 to 12 microM and 334 microM, respectively. The enzyme also accepted other aromatic aldehydes as substrates, whereas aliphatic aldehydes were not accepted. The NH2-terminal amino acid sequence of CALDH was determined in order to clone the encoding gene (calB). The corresponding nucleotide sequence was localized on a 9.4-kbp EcoRI fragment (E94), which was subcloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The partial sequencing of this fragment revealed an open reading frame of 1,446 bp encoding a protein with a relative molecular weight of 51,822. The deduced amino acid sequence, which is reported for the first time for a structural gene of a CALDH, exhibited up to 38.5% amino acid identity (60% similarity) to NAD+-dependent aldehyde dehydrogenases from different sources.

Identification and disruption of the gene encoding the K+-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae

Abstract: The identity of the gene encoding the mitochondrial K+-activated acetaldehyde dehydrogenase (K+-ACDH) of Saccharomyces cerevisiae has been confirmed. The gene is situated on the right arm of chromosome XV, bears the systematic name YOR374w and the deduced product shows significant homology to other members of the S. cerevisiae aldehyde dehydrogenase (ALDH) family. YOR374w has now been assigned the gene name ALD7. The N-terminal amino acid sequences of K+-ACDHs purified from several diverse strains of S. cerevisiae were determined, and found to have 81–100% identity in alignments with the product of ALD7. Haploid mutants containing a deletion of ALD7 were constructed and, in these strains, the K+-ACDH was not detectable under any growth conditions examined. The activity of the Mg2+-activated acetaldehyde dehydrogenase (Mg2+-ACDH), encoded by ALD6, remained at wild-type levels in the mutants. Growth on glucose was not affected in the mutants lacking ALD7 (in contrast to the behaviour of ald6 mutants), whereas growth on ethanol was severely impaired. This observation, together with previous work by our group, shows that both the Mg2+- and K+-ACDHs are required for growth on ethanol, whilst only the former plays a role during growth on glucose.

Genetic polymorphism of CYP2E1, ADH2, and ALDH2 in Mexican-Americans.

Abstract: The major enzymes involved in the metabolism of ethanol are alcohol dehydrogenases (ADH) and aldehyde dehydrogenase (ALDH). Some of the isozymes of ADH are expressed polymorphically. Studies investigating a causal link between ADH expression and alcoholic liver disease (ALD) have so far produced conflicting results. The cytochrome P450 2E1 (CYP2E1) represents a second enzyme that can metabolize ethanol. Although normally a minor route of metabolism, its role in chronic alcoholics may be proportionately greater than in nonalcoholics because CYP2E1 is inducible by ethanol. An Rsa I restriction fragment length polymorphism (RFLP) in the 5'-flanking region of the CYP2E1 gene has been identified. Studies have shown that the mutant allele demonstrates greater transcriptional rate, protein level, and enzyme activity when compared with the wild-type allele. The association between the Rsa I site polymorphism and ALD has been reported. In this report, we examined the genotypes of ADH2(2), ALDH2(2), and CYP2E1 in a group of healthy subjects of Mexican-American descent. The ADH2(2) and ALDH2(2) frequencies are 6% and 0%, respectively, which are similar to those which have been reported for Caucasians. In contrast, the Rsa I allele frequency of the CYP2E1 gene is 16%, which is significantly higher than in Caucasians. The high RsaI allele frequency found in Mexican-Americans suggests that it might play a role in the development of ALD in this rapidly growing minority population where ALD is common.

Enhanced Expressions of Glucose-6-Phosphate Dehydrogenase and Cytosolic Aldehyde Dehydrogenase and Elevation of Reduced Glutathione Level in Cyclophosphamide-Resistant Human Leukemia Cells

Abstract: ABSTRACT: Elevation of activity and mRNA level of a cytosolic aldehyde dehydrogenase-1 (ALDH1), which oxidizes aldophosphamide, was previously observed in a cyclophosphamide-resistant murine leukemia cell line. However, changes in other enzyme(s) which may detoxify the drug or produce anti-alkylating agent(s), have not been examined. The human leukemia cell line, K562, was made 30-fold resistant against 4-hydroperoxyphosphamide (4HC) by exposing the cells to increasing concentrations of the drug. Resistance against cisplatin was also increased by about 3-fold. Activities of glucose-6-phosphate dehydrogenase (G6PD) and ALDH1 were elevated more than 7-fold in the resistant cells. The mRNA level of the two enzymes was also proportionally elevated. The concentration of reduced glutathione (GSH) was higher in the resistant cells (i.e., 21.1 versus 4.68 nmole per 10 6 cells), while activities of γ-glutamylcysteine synthetase and glutathione synthetase, and the expressions of other human ALDH genes were not increased in the resistant cells. These findings suggest that the acquired resistance against 4HC is a consequence of transcriptional activation of two genes, i.e., one encoding the G6PD, a major enzyme regenerating anti-alkylating GSH, and the other encoding ALDH1, which has a high activity for oxidation of aldophosphamide derived from 4HC.

Identification of the Human P-450 Enzymes Responsible for the Sulfoxidation and Thiono-Oxidation of Diethyldithiocarbamate Methyl Ester: Role of P-450 Enzymes in Disulfiram Bioactivation

Abstract: Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.

Drosophila melanogaster alcohol dehydrogenase: mechanism of aldehyde oxidation and dismutation.

Abstract: Drosophila alcohol dehydrogenase (Adh) catalyses the oxidation of both alcohols and aldehydes. In the latter case, the oxidation is followed by a reduction of the aldehyde, i.e. a dismutation reaction. At high pH, dismutation is accompanied by a small release of NADH, which is not observed at neutral pH. Previously it has been emphasized that kinetic coefficients obtained by measuring the increase in A340, i.e. the release of NADH at high pH is not a direct measure of the aldehyde oxidation reaction and these values cannot be compared with those for alcohol dehydrogenation. In this article we demonstrate that this is not entirely true, and that the coefficients phiB and phiAB, where B is the aldehyde and A is NAD+, are the same for a dismutation reaction and a simple aldehyde dehydrogenase reaction. Thus the substrate specificity of the aldehyde oxidation reaction can be determined by simply measuring the NADH release. The coefficients for oxidation and dehydrogenation reactions (phi0d and phiAd respectively) are complex and involve the constants for the dismutation reaction. However, dead-end inhibitors can be used to determine the quantitative contribution of the kinetic constants for the aldehyde oxidation and reduction pathways to the phi0d and phiAd coefficients. The combination of dead-end and product inhibitors can be used to determine the reaction mechanism for the aldehyde oxidation pathway. Previously, we showed that with Drosophila Adh, the interconversion between alcohols and aldehydes followed a strictly compulsory ordered pathway, although aldehydes and ketones formed binary complexes with the enzyme. This raised the question regarding the reaction mechanism for the oxidation of aldehydes, i.e. whether a random ordered pathway was followed. In the present work, the mechanism for the oxidation of different aldehydes and the accompanying dismutation reaction with the slow alleloenzyme (AdhS) from Drosophila melanogaster has been studied. To obtain reliable results for the liberation of NADH during the initial-rate phase, the reaction was measured with a sensitive recording filter fluorimeter, and the complexes formed with the different dead-end and product inhibitors have been interpreted on the basis of a full dismutation reaction. The results are only consistent with a compulsory ordered reaction mechanism, with the formation of a dead-end binary enzyme-aldehyde complex. Under initial-velocity conditions, the rate of acetate release was calculated to be larger than 2.5 s-1, which is more than ten times that of NADH. The substrate specificity constant (kcat/Km or 1/phiB) with respect to the oxidation of substrates was propan-2-ol>ethanol>acetaldehyde>trimethylacetaldehyde.

Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus

Abstract: The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases.

Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase

Abstract: The prototypic aromatic C-nitroso compound, nitrosobenzene (NB), was shown previously to mimic the effect of nitroxyl (HN=O), the putative active metabolite of cyanamide, in inhibiting aldehyde dehydrogenase (AlDH). To minimize the toxicity of NB in vivo, pro-prodrug forms of NB, which were designed to be bioactivated either by an esterase intrinsic to AlDH or the mixed function oxidase enzymes of liver microsomes, were prepared. Accordingly, the prodrug N-benzenesulfonyl-N-phenylhydroxylamine (3) was further latentiated by conversion to its O-acetyl (1a), O-methoxycarbonyl (1b), O-ethoxycarbonyl (1c), and O-methyl (2) derivatives. Similarly, pro-prodrug forms of nitroxyl were prepared by derivatization of the hydroxylamino moiety of methanesulfohydroxamic acid with N, O-bis-acetyl (7a), N,O-bis-methoxycarbonyl (7b), N, O-bis-ethoxycarbonyl (7c), and N-methoxycarbonyl-O-methyl (7d) groups. It was expected that the bioactivation of these prodrugs would initiate a cascade of nonenzymatic reactions leading to the ultimate liberation of NB or nitroxyl, thereby inhibiting AlDH. Indeed, the ester pro-prodrugs of both series were highly active in inhibiting yeast AlDH in vitro with IC50 values ranging from 21 to 64 microM. However, only 7d significantly raised ethanol-derived blood acetaldehyde levels when administered to rats, a reflection of the inhibition of hepatic mitochondrial AlDH-2.