Flavin dependent enzymes

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- 

A contrasting mode of flavoprotein reactivity with an acetylenic inactivator occurs in the reaction of 2-hydroxy-3-butynoate (13, Fig. 15) with a number of a-hydroxy acid oxidizing enzymes. This process is exemplified by the inactivation of L-lactate oxidase from Mycobacterium smegmatis, an enzyme which catalyzes the oxidative decarboxylation of lactate to yield acetate, carbon dioxide, and water (Walsh, 1979, p. 408). Incubation of 13 with lactate oxidase leads to inactivation of the enzyme with a partition ratio that varies from 110 in the 

The enzyme D-lactate dehydrogenase from Megasphaera elsdenii catalyzes the oxidation of D-lactate to pyruvate, with an electron-transferring flavoprotein serving as the ultimate oxidant. Its reaction is similar to the first step of the lactate oxidase reaction, but the two enzymes use enantiomeric substrates, leading to the proposal that the two enzymes utilize similar mechanisms but bind their substrates in opposite orientations (Ghisla et al., 1976). Incubation of o-lactate dehydrogenase with d-13 leads to enzyme inactivation with a partition ratio of 5 (Olson et al., 1979). A novel pink chromophore formed concomitantly 

Monoamine oxidase is also susceptible to inactivation by olefins such as ally-lamine. An isotope effect of 2.3S on inactivation by [l- Halallylamine and formation of a reduced flavin spectrum are consistent with monoamine oxidase-catalyzed oxidation of the compound (Rando and Eigner, 1977). Because the 

1- propyne. Reexamination of the inactivation process by Silverman and coworkers (1985) revealed that the flavin is rapidly reoxidized on denaturation of the enzyme, indicating that the modification must reside on the enzyme. Treatment of the modified enzyme with benzylamine leads to recovery of oxidase activity and isolation of products consistent with addition of an amino acid residue to C-3 of propanal. In the mechanism suggested to account for the inactivation process (Fig. 20), the flavin semiquinone radical is in equilibrium with an amino acid radical, allowing modification of the enzyme to occur by radical recombination. 

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Like the examples above, dihydroxyacetanilide epoxidase (DHAE) uses an olefin as the substrate for epoxidation. Its mechanism, however, is fundamentally different from those of cytochrome P450 or flavin-dependent enzymes. Dihydroxyacetanilide is an intermediate in the biosynthesis of the epoxyquinones LL-C10037a, an antitumor agent produced by the actinomycete Streptomyces LL-C10037 [75, 76], and MM14201, an antibiotic produced by Streptomyces MPP 3051 (Scheme 10.20) [77]. The main structural difference between the two antibiotics lies in the opposite stereochemistry of the oxirane ring. 

The straightforward concept for the direct light-driven regeneration of flavin-dependent enzymes has been successfully applied for two representative classes of such enzymes a reductase and a monooxygenase. Therefore, it can be expected that this concept can also be applied to other flavin-dependent enzymes, potentially leading to additional practical catalyst systems for applications in synthetic organic chemistry. 

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. 

JBC(244)2590,76JBC(251)6994>. The compounds monooxygenated by flavin-dependent enzymes include both electrophilic and nucleophilic species. These compounds can be divided into three groups for the convenience of discussion (i) amines and sulfides, (ii) aromatic compounds with electron releasing substituents, and (iii) aldehydes and ketones (Table 3). 

Clearly, the chemistry carried out on model systems for both B12-coenzyme dependent and flavin-dependent enzymes have suggested a number of mechanisms by which the natural systems might function. As with all biomimetic chemistry, the information given by the in vitro studies must be compared with and extended to the in vivo system to assess whether or not the model chemistry bears any relationships to the natural systems. 

Dietary deficiency is relatively widespread, yet is apparently never fatal there is not even a clearly characteristic riboflavin deficiency disease. In addition to intestinal bacterial synthesis of the vitamin, there is very efficient conservation and reutilization of riboflavin in tissues. Flavin coenzymes are tightly enzyme bound, in some cases covalently, and control of tissue flavins is largely at the level of synthesis and catabolism of flavin-dependent enzymes. 

Control over tissue concentrations of riboflavin coenzymes seems to be largely by control of the activity of flavokinase, and the synthesis and catabolism of flavin-dependent enzymes. Almost all the vitamin in tissues is enzyme bound, and free riboflavin phosphate and FAD are rapidly hydrolyzed to riboflavin. If this is not rephosphorylated, it rapidly diffuses out of tissues and is excreted. 

The activities of a variety of flavin-dependent enzymes are depressed in hypothyroidism. They are increased by the administration of thyroxine or triiodothyronine, as a result of increased synthesis of riboflavin phosphate and... 

Most aromatic hydroxylases are either cytochrome- or flavin-dependent enzymes the three enzymes that catalyze hydroxylation of the aromatic amino acids phenylalanine, tyrosine, and tryptophan are apparently unique in... 

The stmcture of PdR (Figure 9) is significantly different from that of AdR although both proteins are related to disulfide reductases and other flavin-dependent enzymes. The... 

Certain CoA thioester using enzymes catalyze reactions at the fS-carbon or other carbons of the acyl group more distant from the thioester functionality. The fatty acid fi-oxidation cycle provides some examples (Fig. 3). Fatty acids 7 enter the cycle by initial conversion to the CoA ester 8, which is then oxidized to the a,P-unsaturated thioester 9 by a flavin-dependent enzyme. Addition of water to the double bond to form the fi-hydroxy thioester 10 is catalyzed by the enzyme crotonase, which is the centerpiece of the crotonase superfamily of enzymes that catalyze related reactions (37), which is followed by oxidation of the alcohol to form the fi-keto thioester 11. A retro-Claisen reaction catalyzed by thiolase forms acetyl-CoA 12 along with a new acyl-CoA 13 having a carbon chain two carbons shorter than in the initial or previous cycle. 

Palfey BA, Massey V. Flavin-dependent enzymes. In Comprehensive Biochemical Catalysis. Sinnott M, ed. 1998. Academic Press, New York, pp. 83-154. 

The substrate for cobalt insertion was found to be the fl,c-diamide 78 generated from hydrogenobyrinic acid 60 by CobB. Remarkably, the superficially simple step of cobalt insertion requires the cooperation of three proteins, CobN, Cobs and CobT, to form a cobalt cheletase system which is ATP-dependent. The product, cob(II)yrinic acid fl,c-diamide, is reduced to the Co(l) state by a flavin dependent enzyme (gene as yet unknown), which was isolated as pure protein. The highly nucleophilic Co(I)-complex was thus ready for adenosylation at... 

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). 

Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science 259, 965-967. 

Recently, laccases found some interest for synthetic application. Laccases are widely distributed in plants and fungi1131. The copper-containing enzymes are some of the few oxidases so far reported to reduce molecular oxygen to water (aside from cytochrome c oxidase and others). This ability was recently exploited in a novel regeneration concept for flavin-dependent enzymes (see Chapter 16.2)[14]. 

A. Palfey, V. Massey, Flavin-Dependent Enzymes. In Comprehensive Biological Catalysis. M. Sinnott (ed), Academic Press, San Diego, London, 1998, Vol. Ill, pp. 83-154. 

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