Changes to the oxidation state of a molecule are frequently carried out as a secondary metabolite is synthesized or modified. The processes are not always completely understood, but the following general features are recognized. The processes may be classified according to the type of enzyme involved and their mechanism of action.
Dehydrogenases remove two hydrogen atoms from the substrate, passing them to a suitable coenzyme acceptor. The coenzyme system involved can generally be related to the functional group being oxidized in the substrate. Thus if the oxidation process is
then a pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+), tends to be utilized as hydrogen acceptor. One hydrogen from the substrate (that bonded to carbon) is transferred as hydride to the coenzyme, and the other, as a proton, is passed to the medium (Figure 2.16). NAD(P)+ may also be used in the oxidations
The reverse reaction, i.e. reduction, is also indicated in Figure 2.16, and may be compared with the chemical reduction process using complex metal hydrides, e.g. LiAlH4 or NaBH4, namely nu-cleophilic addition of hydride and subsequent protonation. The reduced forms NADH and NADPH are conveniently regarded as hydride-donating
Dehydrogenases: NAD+ and NADP+
Dehydrogenases: FAD and FMN
1 1 1 1 1
FMN Figure 2.17
reducing agents. In practice, NADPH is generally employed in reductive processes, whilst NAD+ is used in oxidations.
Should the oxidative process be the conversion
the coenzyme used as acceptor is usually a flavin nucleotide, flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). These entities are bound to the enzyme in the form of a flavoprotein, and take up two hydrogen atoms, represented in Figure 2.17 as being derived by addition of hydride from the substrate and a proton from the medium. Alternative mechanisms have also been proposed, however. Reductive sequences involving flavoproteins may be represented as the reverse reaction in Figure 2.17. NADPH may also be employed as a coenzyme in the reduction of a carbon - carbon double bond.
These oxidation reactions employing pyridine nucleotides and flavoproteins are especially important in primary metabolism in liberating energy from fuel molecules in the form of ATP. The reduced coenzymes formed in the process are normally reoxidized via the electron transport chain of oxidative phosphorylation, so that the hydrogen atoms eventually pass to oxygen giving water.
Oxidases also remove hydrogen from a substrate, but pass these atoms to molecular oxygen or to hydrogen peroxide, in both cases forming water. Oxidases using hydrogen peroxide are termed per-oxidases. Mechanisms of action vary and need not be considered here. Important transformations in secondary metabolism include the oxidation of ortho- and para-quinols to quinones (Figure 2.18), and the peroxidase-induced phenolic oxidative coupling processes (see page 28).
Oxygenases catalyse the direct addition of oxygen from molecular oxygen to the substrate. They are subdivided into mono- and di-oxygenases according to whether just one or both of the oxygen atoms are introduced into the substrate. With mono-oxygenases, the second oxygen atom from O2 is reduced to water by an appropriate hydrogen
OH para-quinol a:
donor, e.g. NADH, NADPH, or ascorbic acid (vitamin C). In this respect they may also be considered to behave as oxidases, and the term 'mixed-function oxidase' is also used for these enzymes. Especially important examples of these enzymes are the cytochrome P-450-dependent mono-oxygenases. These are frequently involved in biological hydroxylations, either in biosynthesis, or in the mammalian detoxification and metabolism of foreign compounds such as drugs, and such enzymes are thus termed 'hydroxylases' . Cytochrome P-450 is named after its intense absorption band at 450 nm when exposed to CO, which is a powerful inhibitor of these enzymes. It contains an iron - porphyrin complex (haem), which is bound to the enzyme, and a redox change involving the Fe atom allows binding and the cleavage of an oxygen atom. Many such systems have been identified, capable of hydroxy-lating aliphatic or aromatic systems, as well as producing epoxides from alkenes (Figure 2.19). In most cases, NADPH features as hydrogen donor.
Aromatic hydroxylation catalysed by mono-oxygenases (including cytochrome P-450 systems) probably involves arene oxide (epoxide) intermediates (Figure 2.20). An interesting consequence of this mechanism is that when the epoxide opens up, the hydrogen atom originally attached to the position which becomes hydroxylated can migrate to the adjacent carbon on the ring. A high proportion of these hydrogen atoms is subsequently retained in the product, even though enolization allows some loss of this hydrogen. This migration is known as the NIH shift, having been originally observed at the National Institute of Health, Bethesda, MD, USA.
hydride migration NIH shift
loss of labelled l| hydrogen
H ^ enolization
loss of labelled l| hydrogen
retention of labelled H hydrogen
retention of labelled H hydrogen
The oxidative cyclization of an ortho-hydroxy-methoxy-substituted aromatic system giving a methylenedioxy group is also known to involve a cytochrome P-450-dependent mono-oxygenase. This enzyme hydroxylates the methyl to yield a formaldehyde hemiacetal intermediate, which can cyclize to the methylenedioxy bridge (the acetal of formaldehyde) by an ionic mechanism (Figure 2.21).
Dioxygenases introduce both atoms from molecular oxygen into the substrate, and are frequently involved in the cleavage of bonds, including aromatic rings. Cyclic peroxides (dioxetanes) are likely to be intermediates (Figure 2.22). Oxidative cleavage of aromatic rings typically employs cat-echol (1,2-dihydroxy) or quinol (1,4-dihydroxy) substrates, and in the case of catechols, cleavage may be between or adjacent to the two hydrox-yls, giving products containing aldehyde and/or carboxylic acid functionalities (Figure 2.22).
Some dioxygenases utilize two acceptor substrates and incorporate one oxygen atom into each. Thus, 2-oxoglutarate-dependent dioxygenases hydroxylate one substrate, whilst also transforming 2-oxoglutarate into succinate with the release of CO2 (Figure 2.23). 2-Oxoglutarate-dependent dioxygenases also require as cofactors
Methylenedioxy groups ch3 o2
nucleophilic attack on © to carbonyl equivalent
formaldehyde hemiacetal methylenedioxy derivative
Dioxygenases | + O2
+ O, dioxetane
cleavage between hydroxyls
cleavage adjacent to hydroxyls
Amine oxidases ho2c
2-oxoglutarate-dependent dioxygenase ho2c co2h
Fe2+ to generate an enzyme-bound iron - oxygen complex, and ascorbic acid (vitamin C) to subsequently reduce this complex.
In addition to the oxidizing enzymes outlined above, those which transform an amine into an aldehyde, the amine oxidases, are frequently involved in metabolic pathways. These include monoamine oxidases and diamine oxidases. Monoamine oxidases utilize a flavin nucleotide, typically FAD, and molecular oxygen, and involve initial dehydrogenation to an imine, followed by hydrolysis to the aldehyde and ammonia (Figure 2.24). Diamine oxidases require a diamine substrate, and oxidize at one amino group using molecular oxygen to give the corresponding aldehyde. Hydrogen peroxide and ammonia are the other products formed. The aminoaldehyde so formed then has the potential to be transformed into a cyclic imine via Schiff base formation.
FAD, O2 NH3
monoamine oxidase o2, h2o nh3, h2o2 h2^^Tnh2 V ^ h2^ ^cho diamine oxidase
The chemical oxidation of ketones by peracids, the Baeyer- Villiger oxidation, yields an ester, and the process is known to involve migration of an alkyl group from the ketone (Figure 2.25). For comparable ketone ^ ester conversions known to occur in biochemistry, cytochrome-P-450- or FAD-dependent enzymes requiring NADPH and O2 appear to be involved. This leads to formation of a peroxy - enzyme complex and a mechanism similar to that for the chemical Baeyer - Villiger oxidation may thus operate. The oxygen atom introduced thus originates from O2 .
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