Flavin dependent oxidase

Xanthine oxidoreductase and aldehyde oxidase represent a distinct class of flavin-dependent oxidases. They both dehydrogenate andhydroxylate the substrate. However, unlike the mixed-function oxidases (Section 7.3.8), the oxygen introduced into the substrate by these enzymes is derived from water, and the role of molecular oxygen is in the reoxidation of the reduced flavin. Among other reactions, aldehyde oxidase is important in the oxidation of A( -methyl... 

Figure 5 Illustration of possible partial reaction cycles of some copper- and flavin-dependent oxidase enzymes, (a) Copper amine oxidase 30, 31 (b) galactose oxidase (32) (c) catechol oxidase (10) (d) multicopper oxidases (10) (e) flavin oxidases (30) (f) cytochrome c oxidase (38).

Mechanistically, it has been proposed that the reaction proceeds predominantly via epoxidation of the aromatic species 28, which leads to unstable arene-oxides 29-31 (equation 47). Rearrangement of the arene-oxides 29-31 involving the migration of a hydride anion (NIH-shift) forms the phenolic product 32 or 33. Alternative flavin-dependent oxidases have been proposed to involve a hydroperoxide intermediate. ... 

Flavin-dependent oxidases and dehydrogenases mediate the net two-electron oxidation of their respective substrates with the formation of a reduced flavin intermediate. In a subsequm oxidative half-reaction, oxidases transfer two electrons to molecular oxygen, whereas dehydrogenases utilize one-electron red-... 

The phosphorylated and non-phosphorylated forms of vitamin Bg have various physical and chemical properties. Vitamin Bs in the form of pyridoxal-5 -phosphate (PLP) and to a lesser extent, pyridoxamine-5 -phosphate (PMP), functions as a coenzyme in over 100 enzymatic reactions. All the forms of vitamin Be possess vitamin activity because they can be converted in vivo to pyridoxal. PN, PM and PL are converted to 5 -phosphate by a single kinase enzyme which in the brain and liver is most active with zinc. PNP and PMP are then converted to PLP by flavin dependent oxidase this is the reason why vitamin B2 deficiency causes a fall in available PLP (Holman 1995). Human cells can synthesize PLP from three vitamers via the Bg salvage pathway but cannot synthesize PLP de novo and must obtain it from dietary sources. 

Oxidation and Reduction.—Shinkai and co-workers have developed micellar models for flavin-dependent oxidases, which operate through the trapping of reactive carbanions. Thus 4-chlorobenzoylformic acid reacts with cyanide ion in aqueous solution to give 4,4 -dichlorobenzoin (72%) together with a small quantity of 4-chlorobenzoic acid (2.4%) formed by aerial oxidation of the carbanion. In the presence of a cationic surfactant micelle, the yield of oxidation product is greatly increased and there is a further rate acceleration in the presence of the hydrophobic flavin (34) since the mechanism shown (Scheme 2) is now strongly favoured. This type of reaction path also accounts for the oxidation of aromatic aldehydes in the same reaction system, and molecular oxygen may replace the flavin but with lower efficiency. The oxidation of nitroethane to acetaldehyde is catalysed by a similar combination of hydrophobic flavin and cationic surfactant and does not occur in their absence. 

An advanced technique for the clean generation of (25) in situ is based on the oxidation of i-glycerol 3-phosphate (35) catalyzed by microbial flavine-dependent glycerol phosphate oxidases (GPO) (Figure 10.19, box) [102]. This method generates... 

In contrast to the flavin-dependent monoamine oxidases, SSAO/VAP-1 has evolved to hydroxylate a tyrosine residue in the active site which is further oxidized to the quinone state by oxygen in the presence of copper ion releasing hydrogen peroxide [28-30]. The primary amine in the substrate (R-NH2, Scheme 1) forms a Schiff-base with the quinone carbonyl group, which through a series of steps ultimately releases the aldehyde product. 

In flavin-dependent monooxygenases, a flavin-oxygen intermediate reacts with the substrate, also producing water in a second step, and requiring cofactors for regeneration of the flavin moiety. The unusual flavoprotein vanillyl-alcohol oxidase (EC 1.1.3.38), in which the flavin moiety is covalently bound, catalyzes the oxidation of p-substituted phenols as well as deamination, hydroxylation and dehydrogenation reactions [10]. 

Monoamine oxidases (MAOs) are mitochondrial membrane enzymes. These flavin-dependent enzymes are responsible for the oxidative deamination of numerous endogenic and exogenic amines (norepinephrine, serotonin, dopamine, etc.). MAO A and B take part in the regulation of these amines in many organs, such as the brain. The essential physiological role of these amines, especially in the central nervous system, has motivated the search for inhibitors of their catabolism in order to enhance the synaptic concentration of neuroamines. 

Flavins are very versatile redox coenzymes. Flavopro-teins are dehydrogenases, oxidases, and oxygenases that catalyze a variety of reactions on an equal variety of substrate types. Since these classes of enzymes do not consist exclusively of flavoproteins, it is difficult to define catalytic specificity for flavins. Biological electron acceptors and donors in flavin-mediated reactions can be two-electron acceptors, such as NAD+ or NADP+, or a variety of one-electron acceptor systems, such as cytochromes (Fe2+/ Fe3+) and quinones, and molecular oxygen is an electron acceptor for flavoprotein oxidases as well as the source of oxygen for oxygenases. The only obviously common aspect of flavin-dependent reactions is that all are redox reactions. 

The latest, and most advanced, technique uses an inexpensive flavine-dependent glycerol phosphate oxidase (GPO, EC 1.1.3.21), found in several microorganisms (Scheme 13), for air oxidation of L-glycerol 3-phosphate 65 to generate DHAP practically quantitatively and in high chemical purity [200]. A separate cofactor regeneration step has become obsolete because the reduced... 

Fig. 7.4 Reactions associated with the thioredoxin system. Thioredoxin is a redox-regulating protein with a redox-active disulfide/dithiol within the conserved active site sequence -Cys-Gly-pro-Cys-. Thioredoxin reductase, a 55 kDa flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin (1) and thioredoxin oxidase (2), a flavin-dependent sulfhydry 1 oxidase that catalyzes the oxidative protein folding with the generation of disulfides...

Averbach, A.Z., Pekel, N.D., Seredenko V. I., Kulikov, A.V. and Gvozdev, R.I., (1995) Flavin-dependent alcohol oxidase from the yeast, Pichiapinus. Biochem. J. 310, 601-604. 

The alcohol dehydrogenases were already described in Chapter 3. These enzymes are cofactor dependent and in the active site hydrogen transfer takes place from NADH or NADPH. In the reverse way they can, however, be applied for the oxidation of alcohols in some cases (see below). Oxidases are very appealing for biocatalytic purposes, because they use oxygen as the only oxidant without the need for a cofactor. Oxidases usually have flavins (glucose oxidase, alcohol oxidase) or copper (examples galactose oxidase, laccase and tyrosinase) in the active site [18]. The mechanism for glucose oxidase (GOD) is denoted in... 

Fig. 4.12 Aerobic oxidation of D-glucose catalyzed by flavin-dependent glucose oxidase.

Polyamine oxidase is a flavin-dependent peroxisomal enzyme involved in the degradation of polyamines to putrescine. This assay measures the conversion of /V -acetylspermidine to putrescine. 

The flavin-dependent mixed-function oxidases include amine N-oxidases and a variety of S-oxidases. They provide an alternative to cytochrome P450-dependent enzymes in the metabolism of xenobiotics. 

The second initiation way for the lipoperoxidation in the organism can be defined as semi-enzymatic or quasi-enzymatic. During this mechanism the O " radicals are generated by enzymes including NAD(P)H-dependent oxidases of mitochondrial and microsomal electron transport chaines, NADPH-dependent oxidase of phagocytes, xanthine oxidase and other flavine oxidases. After the HO formation the oxidation process develops in non-enzymatic way. 

A mechamsm that involves protonation of the a-carbanion/enamine of HEThDP and subsequent hydride transfer, which has been proposed for several flavine-dependent enzymes (Pollegioni et al., 1997), is unlikely since no kinetic solvent isotope effect is evident for this catalytic step (Pig. 16.7). In accordance, after replacement of PAD by 5-carba-5-deaza-FAD, a PAD analog not catalyzing a transfer of single electrons but functioning as hydride acceptor, no reduction is observed by the HEThDP intermediate in pyruvate oxidase from Lactobacillus plantarum (Tittmann et al., 1998). 

---