Aldehyde oxidase substrate specificity

R is an electron-donor substrate such as purine or xanthine and A is an electron acceptor such as 02 or NAD+. It is thought that the in vivo mammalian form of xanthine oxidase uses NAD+ as acceptor and is therefore, more appropriately named xanthine dehydrogenase. No evidence exists for a dehydrogenase form of aldehyde oxidase. The specificities of xanthine oxidase and aldehyde oxidase have been extensively catalogued (96), and the mechanism and properties of these enzymes have been reviewed (97, 98). 

Ohkubo, M., and Fujimura, S. Rat liver N-methyl-nicotinamide oxidase I and II Substrate non-specific enzyme "aldehyde oxidase" and specific enzyme. Biochem Int 4 353-358, 1982. 

Vanillyl alcohol oxidase (VAO) is a flavoenzyme from the ascomycete Penicil-lium simplicissimum that converts a broad range of 4-hydroxybenzyl alcohols and 4-hydroxybenzylamines into the corresponding aldehydes. This large substrate specificity makes it possible to obtain vanillin from two major pathways. 

Aldehyde oxidase catalyzes the oxidation of aldehydes to carboxylic acids by dioxygen, but also catalyzes the hydroxylation of pyrimidines. Despite its rather broad specificity for substrates, it may well be that aldehyde oxidase should be regarded primarily as a pyrimidine hydroxylase. Thus, xanthine oxidase and aldehyde oxidase catalyze the hydroxylation of purines and pyrimidines respectively. The oxygen incorporated into the product comes from water, not 02. The dioxygen serves as the electron acceptor and other oxidizing agents may be used. 

An antennal-specific aldehyde oxidase (AOX) of M. sexta (MsexAOX) was the next identified pheromone-degrading enzyme (Rybczynski el al., 1989). The activity of MsexAOX was visualized on non-denaturing PAGE, and was shown to be antennal specific but present in sensilla of both male and female antennae. MsexAOX was observed as a dimer with a combined estimated molecular mass of 295 kDa. M. sexta uses a multicomponent pheromone consisting exclusively of aldehydes including bombykal (Starratt el al., 1979 Tumlinson el al., 1989, 1994) MsexAOX was shown to degrade bombykal to its carboxylic acid. Both TLC and spectrophotometric assays were established and a variety of substrates and inhibitors were characterized. Making adjustments for the concentrations and volumes within a sensillum lumen, the in vivo half-life of pheromone was estimated at 0.6 msec in the presence of this enzyme (Rybczynski el al., 1989). 

There are a number of molybdenum-containing enzymes, but those that are important in carbon oxidation of xenobiotics are aldehyde oxidase (AO) and xanthine oxidase (XO), also referred to as molybdenum hydroxylases (Figure 10.7). Both enzymes catalyze the oxidation of a wide range of aldehydes and N-heterocycles. The name aldehyde oxidase is somewhat misleading, however, because oxidation of heteroaromatics is more significant. The differences in substrate specificities between... 

Aldehyde oxidase purified from maize coleoptiles is a multicomponent enzyme that contains a molybdenum cofactor, nonheme iron, and flavin adenine dinucleotide (FAD) as prosthetic groups.111 When substrate specificity of the aldehyde oxidase was tested, good activity was detected with IAAld, indole-3-aldehyde, and benzaldehyde among others. The addition of NADP and NADPH did not change the activity. In contrast, in maize endosperm, tryptophan-dependent IAA biosynthesis was dependent on an NADP/NADPH redox system, which may mean that the two tissues of maize are utilizing different pathways or different redox systems for IAA biosynthesis.112... 

Electronic factors and the relative lipophilicity of the molecule probably help to determine the affinity of the substrate for the enzyme as well as turnover properties. It is likely that the fundamental instability of the enzyme has hampered progress in the characterization of human liver aldehyde oxidase. At least in animals, the specific activity of the enzyme is quite dependent on the way the tissue is procured, processed, and stored this may lead to considerable intersample variability. Enzyme instability may at least in part explain why aldehyde oxidase activity from different species is so variable (Duley et al. 1985). However, it is likely that in addition to intrinsic differences in stability, the determination of aldehyde oxidase activity for a given substrate in various tissue preparations is dependent on the analytical methodology employed to assay the enzyme and the likelihood of the presence of different forms of the enzyme that possess distinct substrate specificity and kinetic properties (Johns 1967 Beedham 1985). For example, in the... 

Beedham, C., Critchley, D. J. P., and Ranee, D. J. (1995) Substrate specificity of human liver aldehyde oxidase toward substituted quinazolines and phthalazines a comparison with hepatic enzyme from guinea pig, rabbit, and baboon. Arch. Biochem. Biophys. 319 (2), 481-490. 

There are probably more publications relating to xanthine oxidase than to any other enzyme studied, certainly more than those pertaining to aldehyde oxidase. This is presumably because the former enzyme is easily accessible from cow s milk rather than from animal tissue. It is not the purpose of this review to include all the data amassed on xanthine oxidase, as this has been fully covered in recent reviews [8, 12, 13]. Furthermore, most of our own work has been concerned with aldehyde oxidase. Thus, this report compares the properties of the molybdenum hydroxylases, where possible, in terms of distribution, substrate and inhibitor specificity and mechanism of oxidation. 

Because the molecular properties of aldehyde oxidase and xanthine oxidase do not differ significantly and they contain basically similar substrate-binding sites, it is often assumed that any mechanistic conclusions regarding one enzyme, usually the latter, can be also applied to the other [14, 51]. This may not be a valid assumption to make subtle alterations in the Mo centre may explain varying substrate specificities, but more fundamental modifications of the enzyme molecule may need to be considered to account for the differences in the position of substrate oxidation and, in particular, the rate-determining step. The oxidation of xanthine (3) or lumazine (4) catalysed by xanthine oxidase can be formulated as ... 

In this section are described the important chemical features of those substrates which are oxidized by the molybdenum hydroxylases. Although these enzymes, particularly aldehyde oxidase, also catalyse numerous reductive reactions under anaerobic conditions in vitro, it has not yet been established whether they occur under physiological conditions and there are as yet insufficient examples of any one reduction reaction to permit any conclusions regarding the structure of substrates. Thus, such reactions will not be discussed here (see [11] and references therein). Properties of those inhibitors which bind at the Mo centre and are also substrate analogues will also be included. However, the interaction of inhibitors such as cyanide and arsenite with the molybdenum hydroxylases and the mechanism of action of the specific xanthine oxidase inhibitor, allo-purinol, have been comprehensively described elsewhere [8, 12, 14, 157]. 

The oxidation of substituted benzaldehydes by xanthine oxidase is sterically hindered by bulky substituents at the ortho (o) position (Table 3.5) [167], Increasing the size of the halo-substituent dramatically decreases the oxidation of the o-substituted compound, whereas that of the p-halobenzaldehyde increases due to the increased inductive effect. The positional specificity was not due to electronic effects, because the oxidation rate was also decreased with electron-donating o-substituents. Although the substrates of aldehyde oxidase have not been so rigourously examined, the enzyme does appear to be subject to similar steric considerations, as o-chloro- and o-nitrobenzaldehyde are oxidized at much lower rates than benzaldehyde itself [33]. 

Although aldehyde oxidase and xanthine oxidase differ markedly in their substrate and inhibitor specificities, there is considerable evidence to suggest that binding of both substrates and inhibitors to either enzyme is facilitated by hydrophobic interaction in the enzyme active site. 

Glyoxal oxidase from the white-rot wood-metabolizing basidiomycete Phanerochaete chrysosporium has been characterized as a novel radical—copper oxidase. This enzyme exhibits a wide substrate specificity for oxidation of simple aldehydes to the corresponding carboxylic acids according to Equation (5). 

Xanthina oxidase, xanthine dehydrogenase, Schardtnger enzyme an enzyme of aerobic purine degradation, which catalyses the oxidation of hypoxan-thine to xanthine, and xanthine to uric acid Hypox-anthine + HjO + 62 -> Xanthine -h H2O2 Xanthine + H2O -H O2 -> Uric acid + H2O2. It is a dimeric enzyme, M, 275,000, pH-optimum 4.7, pi 5.35, containing 2 FAD, 2 Mo and 8 Fe (data for the enzyme from milk). The substrate specificity is low it catalyses the oxidation of other purines (e. g. adenine), aU-phatic and aromatic aldehydes, pyrimidines, pteri-dines and other heterocyclic compounds. 

A comparison of xanthine oxidase and aldehyde oxidase was suggested by their functional and structural similarities. Both enzymes catalyze hydroxylation reactions in which water is the source of the hydroxyl group [6,7], both have particle weights around 300,000 [8,9], and both contain FAD, molybdenum, and iron in their internal electron transport chains [7,9]. This report summarizes the results of a comparison of the distributions, substrate specificities and electron acceptor specificities of these two enzymes [10,11] and discusses the possible implications of the findings. 

A comparison of the substrate specificities of bovine milk xanthine oxidase and rabbit liver aldehyde oxidase revealed both similarities and differences [10]. Both enzymes exhibited a preference for heterocycles which contain a condensed-pyrimidine ring system (Figure 3). Of the unsubstituted ring systems studied,... 

The most distinct difference between the substrate specificities of these two enzymes was the effect of the number of C-substituents Both enzymes readily oxidized a variety of unsubstituted and C-monosubstituted condensed-pyrimidines, but only xanthine oxidase readily oxidized any C-disubstituted derivatives. Some examples are given in Figure 4. With aldehyde oxidase, a second C-substituent always markedly decreased substrate activity, while with xanthine oxidase the substrate activity was usually relatively unaffected and, in some cases, actually increased by this substitution. The converse was found with N-substituents Substrate activity with xanthine oxidase was usually obliterated by N-substitution, while with aldehyde oxidase many N-substituents enhanced substrate activity. As an example, the effects of N-methylation on the substrate activity of hypoxanthine with both enzymes are compared in Figure 5. 

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