Kathryn E. Kitson

Sheep liver cytosolic aldehyde dehydrogenase: the structure reveals the basis for the retinal specificity of class 1 aldehyde dehydrogenases.

BACKGROUND . Enzymes of the aldehyde dehydrogenase family are required for the clearance of potentially toxic aldehydes, and are essential for the production of key metabolic regulators. The cytosolic, or class 1, aldehyde dehydrogenase (ALDH1) of higher vertebrates has an enhanced specificity for all-trans retinal, oxidising it to the powerful differentiation factor all-trans retinoic acid. Thus, ALDH1 is very likely to have a key role in vertebrate development. RESULTS . The three-dimensional structure of sheep ALDH1 has been determined by X-ray crystallography to 2.35 A resolution. The overall tertiary and quaternary structures are very similar to those of bovine mitochondrial ALDH (ALDH2), but there are important differences in the entrance tunnel for the substrate. In the ALDH1 structure, the sidechain of the general base Glu268 is disordered and the NAD+ cofactor binds in two distinct modes. CONCLUSIONS . The submicromolar Km of ALDH1 for all-trans retinal, and its 600-fold enhanced affinity for retinal compared to acetaldehyde, are explained by the size and shape of the substrate entrance tunnel in ALDH1. All-trans retinal fits into the active-site pocket of ALDH1, but not into the pocket of ALDH2. Two helices and one surface loop that line the tunnel are likely to have a key role in defining substrate specificity in the wider ALDH family. The relative sizes of the tunnels also suggest why the bulky alcohol aversive drug disulfiram reacts more rapidly with ALDH1 than ALDH2. The disorder of Glu268 and the observation that NAD+ binds in two distinct modes indicate that flexibility is a key facet of the enzyme reaction mechanism.

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin, resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD(+)-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD(+) concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD(+), by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.

The assessment of viability in isolated rat hepatocytes

Isolated rat hepatocytes are used in many metabolic studies, but the viability of these cell preparations is often not adequately established. The present study shows that ATP content is a more reliable index of metabolic viability than trypan blue exclusion. At some of the low trypan blue exclusion levels quoted in the literature, a high percentage of cell preparations is likely to be nonviable by the criterion of ATP content. We suggest that ATP content measured on initial cell preparations and at the end of all incubation procedures is essential for establishing cell viability for metabolic studies on isolated hepatocytes.

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.

The Action of Cytosolic Aldehyde Dehydrogenase on Resorufin Acetate

Aldehyde dehydrogenase is well known to have the ability to act as an esterase as well as to catalyse the oxidation of aldehydes by NAD+. Some workers have been of the opinion that the twin activities of the enzyme are not closely related and occur at separate enzymic sites (see, for example, Motion et al., 1988), whereas we have thought for a number of years that the balance of evidence favours the simpler one-site model (see, for example, Kitson et al., 1991). Recently, the extensive studies of Klyosov et al. (1996) have rekindled the debate; these authors suggest that even within the dehydrogenase activity alone, separate sites are responsible for the oxidation of some substrates (such as acetaldehyde and 6-dimethylamino-2-naphthaldehyde) in human mitochondrial aldehyde dehydrogenase (though not in the cytosolic isozyme).

Reaction between Sheep Liver Mitochondrial Aldehyde Dehydrogenase and A Chromogenic ‘Reporter Group’ Reagent

The cytosolic form of aldehyde dehydrogenase (ALDH-1) has been shown to react slowly with p-nitrophenyl dimethylcarbamate, liberating p-nitrophenoxide and giving an inactive from the enzyme in which Cys-302 carries a -CO-NMe2 label (Kitson et al., 1991) is work led to the idea that a cyclic analogue of the carbamate would constitute a ‘eporter group’ reagent of the type originally envisaged by Burr and Koshland (1964), since the chromophoric p-nitrophenoxide moiety would end up covalently bound within the ene’s active site. The compound in question, namely 3,4-dihydro-3-methyl-6-ni- tro-2-l,3-benzoxazin-2-one or DMNB (see Figure 1) was synthesised and shown to react in the expected way with esterases such such as chymotrypsin (Kitson and Freeman, 1993). Furtheork with ALDH-1 showed that in this case the pKa of the p-nitrophenol reporter group perturbed upwards by about 3 pH units (Kitson and Kitson, 1994). This rather dramatic observation was interpreted to mean that the substrate binding site of ALDH-1 is either very hydrophobic character or contains a negatively charged amino acid sidechain (or conceivably has both characteristics).

Human Class 1 Aldehyde Dehydrogenase

Human Class 1 and Class 2 aldehyde dehydrogenases have been sequenced at both the protein (Hempel et al., 1984, 1985) and DNA level (Hsu et al., 1988, 1989). Studies on the tertiary structure of aldehyde dehydrogenase are in progress (Baker et al., 1995, sheep Class 1; Hurley and Weiner, 1992, beef Class 2), but are not sufficiently advanced to suggest which amino acid residues are important in catalysis. Cys 302 is the only completely conserved cysteine in all known forms of the enzyme (Hempel et al., 1993), and labelling by various substrates and substrate analogues (von Bahr-Lindstrom et al., 1985; Kitson et al., 1991; Pietruszko et al., 1993) has implicated this residue as the probable active site nucleophile. This has been confirmed for the Class 2 enzyme by site-directed mutagenesis (Weiner et al., 1991). In order to establish whether Cys 302 is also the active site nucleophile for Class 1 aldehyde dehydrogenase we decided to carry out mutagenesis at Cys 302. A separate mutant was constructed in which Cys 301, the adjacent residue, was changed to alanine while Cys 302 was left unchanged.

The hunt for a retinal-specific aldehyde dehydrogenase in sheep liver.

During past 7 years, a number of retinal-specific aldehyde dehydrogenases have been identified in rat (RaIDH1, el Akawi and Napoli, 1994; Posch et al, 1992; RalDH2, Wang t al, 1996; RALDH-1, Bhat et al, 1995; Labreque et al, 1993; Labreque et al, 1995) mouse (RALDH-2; Zhao et al, 1996) tissues. In higher mammals such as horse, sheep and human, the major Class 1 aldehyde dehydrogenase (A1DH) is known to oxidise retinal, but it has not been clearly established whether this is the major A1DH that oxidises retinal or whether there is another retinal-specific enzyme as well. The Class 1 A1DH in higher mammals is not specific for retinal; it readily oxidises a wide range of other aldehydes (Klyosov, 1996). For human Class 1 A1DH, previous kinetic studies have yielded varying estimates of the Km for all-trans- retinal. Using a spectrophotometric method giving a single reaction progress curve, Yoshida et al (1992) reported a Km of 0. 06 M, and using a similar method Klyosov (1996) reported a Km of 1. 1 μM. This difference is significant in considering the possible importance of the Class 1 A1DH in retinal metabolism in vivo, since cellular retinal concentrations are likely to be about 0. 1 μM (Yoshidaet al, 1992). The purpose of the current study was two-fold: firstly, to investigate the possibility that retinal-specific AlDHs are present in sheep as well as in rat and mouse sues, and secondly, to carry out more accurate kinetic characterisation of the Class AlDHs from sheep and human with retinal as a substrate.

Covalent Modification of Sheep Liver Cytosolic Aldehyde Dehydrogenase by the Oxidative Addition of Coloured Phenoxazine, Phenothiazine and Phenazine Derivatives

Aldehyde dehydrogenase acts as an esterase towards reactive esters such as p-nitrophenyl acetate, and although the subject of prolonged debate, it is now generally believed that the dehydrogenase and esterase actions of the enzyme involve the same active site and catalytic groups (see Kitson and Kitson, 1996, and references therein). With p -nitrophenyl dimethylcarbamate, the reactivity (compared to the acetate) is so drastically reduced that it takes several hours for this ‘substrate’ to acylate cytosolic aldehyde dehydrogenase (ALDH-l), and the rate of subsequent deacylation is essentially zero (Kitson et al., 1991). Thus the carbamate acts as an active-site-directed irreversible inactivator of the enzyme. It was thought that resorufin dimethylcarbamate (see Figure 1) would react likewise, but since the molar absorptivity of the resorufin anion (69,700) is so much greater than that of p-nitrophenoxide (18,320) (Kitson, 1996), the resorufin carbamate would be a much more sensitive active site titrant than the p-nitrophenyl compound. The results reported and discussed below show that this expectation was not borne out. Resorufin dimethylcarbamate does inactivate ALDH-I, but the chemistry of the reaction is more complicated and interesting than the simple acylation process expected from the previous work with the p-nitrophenyl equivalent; it may be termed ‘oxidative addition’.

Crystallization of Sheep Liver Cytosolic Aldehyde Dehydrogenase in a form Suitable for High Resolution X-Ray Structural Analysis

Aldehyde dehydrogenase (AlDH) is an NAD+-dependent enzyme that is widely distributed through many different species, including both eukaryotes and prokaryotes. In animals, aldehyde dehydrogenases are found in a number of different bodily locations where they exist as several distinct isozymes. These enzymes share the general role of detoxification of aldehydes (Jakoby and Ziegler, 1990) but the individual isozymes show differences in specificity and reactivity that suggest they may have additional, more specialised, roles.