Trevor M. 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.

Intracellular localisation and properties of aldehyde dehydrogenases from sheep liver

Abstract 1. 1. The distribution of aldehyde dehydrogenases in sheep liver was studied. Activity was found in the cytoplasm, mitochondria and microsomes. 2. 2. Cross-contamination of activities from different subcellular fractions, during the isolation procedures used, was shown to be insignificant. Accordingly, the level of aldehyde dehydrogenase activity found in each fraction should reflect the distribution pattern in vivo. 3. 3. Aldehyde dehydrogenases from the cytoplasm and mitochondria were isolated and some of their catalytic properties examined. The results show that the enzymes from the two fractions are not identical.

Studies on possible mechanisms for the interaction between cyanamide and aldehyde dehydrogenase

Abstract Cyanamide is known to interfere with the metabolism of alcohol by decreasing the activity of aldehyde dehydrogenase in vivo , thereby leading to an accumulation of acetaldehyde following the ingestion of ethanol. We have studied various mechanisms for the chemical interaction between aldehyde dehydrogenase and cyanamide (or some of its possible metabolites). Cyanamide was shown to react under physiological conditions with amino and thiol groups, forming guanidino and isothiouronium compounds respectively. However, it was found that the enzyme is not appreciably affected in vitro by high concentrations of cyanamide. Dicyandiamide and the aminoethylisothiouronium ion (AET) also have no effect in vitro . It was postulated that the pathway of in vivo enzyme modification by cyanamide may involve thiourea (formed by breakdown of isothiouronium compounds) and formamidine disulphide (an oxidation product of thiourea). However, although the disulphide is a moderately-effective inactivator of aldehyde dehydrogenase in vitro , administration of thiourea or AET to rats does not result in any significant loss of aldehyde dehydrogenase activity in either the cytoplasm or the mitochondria

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.

Spectrophotometric and Kinetic Studies on the Binding of the Bioflavonoid Quercetin to Bovine Serum Albumin

This work investigates the binding of the bioflavonoid, quercetin, to bovine serum albumin (BSA) by spectrophotometric techniques involving both the conventional and stopped-flow methods. Both the neutral and negatively-charged forms of quercetin bound to BSA with a red shift in the maximal absorption. At high pH values, quercetin was rapidly degraded in an oxygen-dependent process, but this decomposition was substantially slower when the flavonoid was bound to BSA. At pH 7.4, the difference spectrum of quercetin with and without BSA was maximal at 425 nm; this wavelength can be conveniently used to monitor the extent and speed of binding. Spectrophotometric studies with a range of equimolar mixtures of quercetin and BSA at pH 7.4 suggest the binding was maximal when the concentration was 10 μM. It is postulated that the binding site of BSA for quercetin was less available at higher protein concentrations, perhaps because of conformational change or self-association. The rate of spectrophotometric change when quercetin bound to BSA was fairly slow; the process was not quite complete within 45 seconds and was biphasic. When a pre-mixed equimolar mixture of BSA and quercetin was diluted with an equal volume of the buffer, there was a surprising further increase in absorbance at 425 nm (rather than the fall anticipated if the binary complex were to dissociate). It is concluded that, upon dilution, the effective concentration of BSA’s binding site increased, providing more scope for quercetin to bind.

Synthesis of methyl 2- and 4-pyridyl disulfide from 2- and 4-thiopyridone and methyl methanethiosulfonate.

In 1982, methyl 2-pyridyl disulfide was reported as a new reagent for the titration of thiol groups in peptides and proteins and for their temporary blocking with the thiomethyl group [T. Kimura et al. (1982) Anal. Biochem. 122, 274-282]. We have synthesized this compound (and its 4-pyridyl isomer) by a rapid and convenient procedure which is preferable to that in the original report. Our method involves the thiomethylation of the appropriate thiopyridone by methyl methanethiosulfonate.

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.