On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde dehydrogenase [Abriola, D. P., Fields, R., MacKerell, A. D., Jr., & Pietruszko, R. (1987) Biochemistry 26, 5679-5684]. To study its role, the residue in human liver mitochondrial (class 2) aldehyde dehydrogenase was mutated to an aspartate, a glutamine, or a lysine, and the enzyme was expressed in Escherichia coli. The mutations did not affect the Km values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed native enzyme; the mutant enzymes had less that 0.4% of the specific activity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady state phase of the reaction. The activity of the mutant enzymes could not be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The Kd for NADH was essentially identical for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activity of the recombinantly expressed native enzyme. Pre-steady state analysis showed that there was no burst of NADH formation in the dehydrogenase reaction or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a general base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or hydride transfer step.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis.

http://www.jbc.org/content/269/19/13854.full.pdf

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase : a model to study human (oriental type) class 2 aldehyde dehydrogenase

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.

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

The high resolution 2-D protein gel electrophoresis technique combined with MALDI-TOF MS and a recently developed fluorescence-based thiol modification assay were used to investigate the cellular response of Staphylococcus aureus to oxidative stress. Addition of hydrogen peroxide, diamide, and the superoxide generating agent paraquat to exponentially growing cells revealed complex changes in the protein expression pattern. In particular, proteins involved in detoxification, repair systems, and intermediary metabolism were found to be up-regulated. Interestingly, there is only a small overlap of proteins induced by all these stressors. Exposure to hydrogen peroxide mediated a significant increase of DNA repair enzymes, whereas treatment with diamide affected proteins involved in protein repair and degradation. The activity of proteins under oxidative stress conditions can be modulated by oxidation of thiol groups. In growing cells, protein thiols were found to be mainly present in the reduced state. Diamide mediated a strong increase of reversibly oxidized thiols in a variety of metabolic enzymes. By contrast, hydrogen peroxide resulted in the reversible oxidation especially of proteins with active site cysteines. Moreover, high levels of hydrogen peroxide influenced the pI of three proteins containing cysteines within their active sites (GapA1, AhpC, and HchA) indicating the generation of sulfinic or sulfonic acid by irreversible oxidation of thiols.

Proteomic analysis of antioxidant strategies of Staphylococcus aureus: diverse responses to different oxidants.

http://www.jbc.org/content/280/34/30550.full.pdf

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant.

The amino acid sequences of both human class 1 and 2 aldehyde dehydrogenase (ALDH) and the sequences of the genes coding for them are known. Based on this sequence data, we designed primers and isolated the full-length cDNAs encoding both isozymes from a human liver mRNA pool. cDNAs were subcloned in the plasmid pT7-7 and expressed in Escherichia coli with a yield of approximately 3 mg ALDH protein/liter of cell culture, although only one-third of the enzyme was soluble. The soluble recombinantly expressed ALDHs were purified to homogeneity using a hydroxyacetophenone-Sepharose affinity column. The mitochondrial isozyme had a subunit molecular weight of 55 kDa, an isoelectric point of 4.9, and a specific activity of 1.10 units/mg, which were in good agreement with that from the native enzyme. The expressed cytosolic isozyme had the same subunit molecular weight (55 kDa) and pI (5.4) as that reported for the native enzyme and had a specific activity of 0.26 units/mg. The expressed mitochondrial isozyme could be recognized by antibodies raised against rat mitochondrial ALDH, whereas the cytosolic isozyme could be recognized by antibody raised against horse cytosolic ALDH.

Cloning and Expression of the Full‐Length cDNAS Encoding Human Liver Class 1 and Class 2 Aldehyde Dehydrogenase

Mammalian aldehyde dehydrogenase was first purified to homogeneity in our laboratory (Feldman and Weiner, 1972a). The enzyme, isolated from horse liver, was found to be an esterase as well as a non—specific NAD—dependent dehydrogenase. At that time we did not know that the enzyme we were studying (pI=5) was from mitochondria. We found a second minor form of the enzyme with a pI=6; later it was shown that this was the cytosolic form of the enzyme. During our initial attempts to purify the mitochondrial isoenzyme we found a third form with a pI value of 4.8. It had kinetic properties identical with those of the pI 5.0 enzyme form, but it was less stable when heated at 520C. As we became more experienced with isolating the enzyme we failed to identify the low pI form. Though never proven, we felt that this was a degraded form of the mitochondrial isoenzyme.

Site directed mutagenesis to probe for active site components of liver mitochondrial aldehyde dehydrogenase.

It has generally been accepted that aldehyde dehydrogenase functions through covalent catalysis where a nucleophilic amino acid residue attacks the carbonyl of the aldehyde substrate (Weiner et al., 1982). Since the first purification on ALDH by Feldman and Weiner in 1972, it has been argued that the active site nucleophile was a cysteine residue. The identity of this specific cysteine in the primary structure of the enzyme however, has not been unequivocally determined. Recently it has been suggested that the nucleophile is not cysteine but is serine (Loomis et al., 1990). A similar adduct could be formed with either amino acid; a thiohemiacetal in the former, a hemiacetal in the latter. The intermediate would then be oxidized in the presence of NAD to produce a thioacyl or an acyl intermediate respectively, as illustrated in Fig. 1.

Probing the Active Site of Aldehyde Dehydrogenase by Site Directed Mutagenesis

We have recently cloned and expressed the cDNA coding for rat and human liver mitochondrial class 2 aldehyde dehydrogenase (Farres et al., 1989; Jeng and Weiner, 1991, Zheng et al., submitted). The recombinantly expressed enzymes were found to have properties virtually identical to those of the native enzyme. Having an expression system has allowed us to perform site directed mutagenesis to study the role of amino acid residues such as glutamate 487, histidine 235, cysteine 302, and serine 74, to mention just a few. Though we could isolate the enzyme using conventional procedures (Jeng and Weiner, 1991) or a hydroxyacetophenone-Sepharose affinity matrix (Ghenbot and Weiner, submitted) the amount of enzyme produced was very low. Using the plasmid pT7–7 others have reported that as much as 20% of the protein expressed was from their cDNA (see Marston, 1986 for a review) while we could obtain just 1–2 mg of ALDH per liter of cultured cells using the same system. In order for us to study chemical and physical properties of the recombinantly expressed enzymes it will be necessary for us to produce much more enzyme.

Chao-Feng Zheng

Papers

Cloning and Expression of the Full‐Length cDNAS Encoding Human Liver Class 1 and Class 2 Aldehyde Dehydrogenase

Site directed mutagenesis to probe for active site components of liver mitochondrial aldehyde dehydrogenase.

Probing the Active Site of Aldehyde Dehydrogenase by Site Directed Mutagenesis

Attempts to Increase the Expression of Rat Liver Mitochondrial Aldehyde Dehydrogenase in E. coli by Altering the mRNA