Reductive enzymes aldehyde oxidase

The first indication of an essential metabolic role for molybdenum was obtained in 1953, when it was discovered that xanthine oxidase, important in purine metabolism, was a metalloenzyme containing molybdenum. Subsequently the element was shown to be a component of two other enzymes, aldehyde oxidase and sulphite oxidase. The biological functions of molybdenum, apart from its reactions with copper (see p. 123), are concerned with the formation and activities of these three enzymes. In addition to being a component of xanthine oxidase, molybdenum participates in the reaction of the enzyme with cytochrome C and also facilitates the reduction of cytochrome C by aldehyde oxidase. 

A major class of enzymes that catalyze oxidation-reduction reactions. This class includes dehydrogenases, reductases, oxygenases, peroxidases, and a few synthases. Examples include alcohol dehydrogenase (EC 1.1.1.1), aldehyde oxidase (EC 1.2.3.1), orotate reductase (EC 1.3.1.14), glutamate synthase (EC 1.4.1.14), NAD(P) transhydrogenase (EC 1.6.1.1), and glutathione peroxidase (EC 1.11.1.9). 

Aldehydes and Ketones. Many metabolic routes are possible, including both oxidation and reduction. However, oxidations are more common. Aldehydes are very susceptible to oxidation, which is catalyzed by various enzymes including aldehyde oxidase and aldehyde dehydrogenase this oxidation yields a carboxylic acid. Ketones, on the other hand, tend to be stable to oxidation. Conversely, aldehydes are seldom metabolized by reduction. Ketones, however, frequently undergo reduction to a secondary alcohol this is particularly true for a,P-unsaturated ketones. 

The cofactors of both xanthine and aldehyde oxidases belong to the LMoVI(S)(0) subfamily (see Section IV). However, inactive dioxo forms, LMovi(0)2, of both xanthine and aldehyde oxidase are known. These dioxo forms do not catalyze oxidation of the respective substrates of these enzymes. The Mov/Molv redox potential for the inactive bis(oxido) form of xanthine oxidase differs from the oxido-sulfido form by -30 mV (bovine xanthine oxidase) and -lOOmV (chicken liver xanthine oxidase) [91]. Although the difference is small, given the xanthine/uric acid reduction potential (-360 mV), it is possible that the Mov/MoIV couple (-433 mV) of the chicken-liver xanthine oxidase bis(ox-ido) form impedes the effective oxidation of xanthine for redox reasons alone. However, the bis(oxido) form of bovine xanthine oxidase (with a reduction potential of -386 mV) should be able to oxidize xanthine, since the redox potential, and hence the thermodynamic driving force, is sufficient for activity [91,92,99]. As substrate oxidation does not occur, the chemical differences between the bis(oxido) and oxido-sulfido (Movl) forms must be critical to the dramatic difference in activity (see Section VI.E.l). 

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]. 

Aldehyde oxidases (AO) are also molybdenum-containing enzymes that, like XO, exist as homodimers of 300 Kdaltons. It is presumed that they behave mechanistically similarly to XO. Both AO and XO can mediate reductive reactions through the transfer of electrons from FADH2 to oxidized xenobiotic. For example, zonisamide can be reduced by AO to 2-sulfamoylacetylphenol. 

For sulfoxides/sulfides, oxidation is catalyzed by cytochrome P-450 and flavin monooxygenases, whereas the reductive metabolism is catalyzed by aldehyde oxidase and/or thiotedocin-linked enzymes. The fiver as well as the gut and bacterial flora are potential sites for the formation of sulfide metabolites. 

Enzymes catalysing reductions are also found in liver microsomes, e.g. azo-benzene reductase and nitroreductase. Oxidation reactions, which are not due to cytochrome P-450 are catalysed by hexahydrobenzoate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase, and aldehyde oxidase. Several amines are oxidised by monoamine oxidase or diamine oxidase. 

Metallo-Flavoproteins. As was mentioned in the case of cytochrome reductase, enzymes are known that contain metal cofactors in addition to flavin. These are called metallo-flavoproteins. The presence of metals introduces complexity into the reaction, since the metals involved, iron, molybdenum, copper, and manganese, all exist in at least two valence states and can participate in oxidation-reduction reactions. The enzymes known to be metallo-flavoproteins include xanthine oxidase, aldehyde oxidase, nitrate reductase, succinic dehydrogenase, fatty acyl CoA dehydrogenases, hydrogenase, and cytochrome reductases. Before these are discussed in detail some physical properties of flavin will be presented. 

Very early reports described the reduction of nitro substituents by liver xanthine-oxidase (11), an enzyme located in the cytosol. In the microsomal fraction of the rat liver, nitro reduction is catalyzed by a system of cytochrome P450 in combination with NADPH-cyto-chrome-P450-reductase, as was described, for example, by Harada and Omura (19). Other enzymes that have been shown to reduce nitroaromatic compounds include aldehyde oxidase (51), dihydrolipoic amide dehydrogenase (47), cytochrome reductase (31), and dia-phorases (24). 

Xanthine oxidase (XO) was the first enzyme studied from the family of enzymes now known as the molybdenum hydroxylases (HiUe 1999). XO, which catalyzes the hydroxylation of xanthine to uric acid is abundant in cow s milk and contains several cofactors, including FAD, two Fe-S centers, and a molybdenum cofactor, all of which are required for activity (Massey and Harris 1997). Purified XO has been shown to use xanthine, hypoxan-thine, and several aldehydes as substrates in the reduction of methylene blue (Booth 1938), used as an electron acceptor. Early studies also noted that cyanide was inhibitory but could only inactivate XO during preincubation, not during the reaction with xanthine (Dixon 1927). The target of cyanide inactivation was identified to be a labile sulfur atom, termed the cyanolyzable sulfur (Wahl and Rajagopalan 1982), which is also required for enzyme activity. 

There is no valid interpretation for the activation by OJ and by hexacyano-ferrate(III), although they fitted nicely in a reaction scheme with Cu(III) as the active species In the oxidation of an alcohol to an aldehyde Cu(III) would be reduced to Cu(I). In the subsequent reaction of Cu(I) with Oj, Cu(II)Oj was considered an intermediate yielding Cu(III) and H O. This intermediate would be in a reversible equilibrium with OJ and with the resting Cu(II)-enzyme. This resting enzyme would be oxidized by hexacyanoferrate(III) to the active Cu(III) species. There was unfortunately no indication in X-ray absorption measurements for the formation of Cu(III) with hexacyanoferrate(III) and the resting enzyme . EPR measurements indicated that Cu(II) was present in the active enzyme It was not possible, moreover, to detect Oj by the reduction of Fe(III)-cytochrome c in a galactose oxidase — galactose system... 

Enzymes that are glycosylated (i.e., HRP and glucose oxidase) may be oxidized according to the following method to produce aldehyde groups for reductive amination coupling to an antibody molecule. 

Although molybdenum and tungsten enzymes carry the name of a single substrate, they are often not as selective as this nomenclature suggests. Many of the enzymes process more than one substrate, both in vivo and in vitro. Several enzymes can function as both oxidases and reductases, for example, xanthine oxidases not only oxidize purines but can deoxygenate amine N-oxides [82]. There are also sets of enzymes that catalyze the same reaction but in opposite directions. These enzymes include aldehyde and formate oxidases/carboxylic acid reductase [31,75] and nitrate reductase/nitrite oxidase [83-87]. These complementary enzymes have considerable sequence homology, and the direction of the preferred catalytic reaction depends on the electrochemical reduction potentials of the redox partners that have evolved to couple the reactions to cellular redox systems and metabolic requirements. 

As with xanthine oxidase, the sulfido ligand of the active form of aldehyde oxidoreductase is readily replaced by an oxido ligand to yield a cofactor with a structure that resembles that of oxidized sulfite oxidase and assimilatoiy nitrate reductase. Both x-ray and EXAFS data are available for the bis(oxido) form, and, with the exception of the oxido replaced sulfido ligand, few changes are obvious in the overall structure of the oxidized form of the desulfo cofactor. Upon reduction of the enzyme the oxido ligand is presumably reduced to hydroxido, an observation that is supported by EPR data for the Mov state. 

Enantioselective oxidation of racemic alcohols as well as reduction of racemic ketones and aldehydes have been widely applied to obtain optically active alcohols.25 27 The enzymes catalyzing these reactions are alcohol dehydrogenase, oxidases, and reductases etc. Coenzymes (NADH, NADPH, flavine etc) are usually necessary for theses enzymes. For example, for the oxidation of alcohols, NAD(P)+ are used. The hydride removed from the substrate is transferred to the coenzyme bound in the enzyme, as shown in Figure 24. There are four stereochemical patterns, but only three types of the enzymes are known. 

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