Flavoprotein monooxygenase

Phenazines — The phenazines are biosynthesized by the shikimic acid pathway, through the intermediate chorismic acid. The process was studied using different strains of Pseudomonas species, the major producers of phenazines. The best-known phenazine, pyocyanine, seems to be produced from the intermediate phenazine-1-carboxylic acid (PCA). Although intensive biochemical studies were done, not all the details and the intermediates of conversion of chorismic acid to PCA are known. In the first step, PCA is N-methylated by a SAM-dependent methyltransferase. The second step is a hydroxylative decarboxylation catalyzed by a flavoprotein monooxygenase dependent on NADH. PCA is also the precursor of phenazine-1-carboxamide and 1-hydroxyphenazine from Pseudomonas species. - - ... 

The ability to grow at the expense of 4-hydroxy- and 3,4-dihydroxybenzoate has been nsed for the classification of medically important yeasts inclnding Candida parapsilosis (Cooper and Land 1979). This organism degrades these snbstrates by oxidative decarboxylation, catalyzed by a flavoprotein monooxygenase (Eppink et al. 1997). 

Although flavoprotein monooxygenases form a separate class, formally they can be considered to react according to Equation (4) as far as the redox states involved in catalysis are concerned. 

Bacterial flavoprotein monooxygenase (cyclohexanone oxygenase) has been used for the oxidation and rearrangement of allyl selenides, but the products have not been analyzed for enantiomer composition10,11. 

Flavoprotein monooxygenases mainly use NAD(P)H as electron donor and insert one atom of molecular oxygen into then-substrates. Oxygen activation of flavoprotein monooxygenases involves the (transient) stabilization of a flavin C4a-(hydro) peroxide. This species performs either a nucleophilic or electrophilic attack on the substrate (Fig. 6). Oxygenation reactions catalyzed by flavoprotein monooxygenases include hydroxyla-tions, epoxidations, Baeyer-Villiger oxidations, and sulfoxida-tions (43). 

Figure 6 General mechanism for flavoprotein monooxygenases. With Baeyer-Villiger monooxygenases (nucleophilic oxygenation), NADP+ stays bound during the entire reaction cycle.

Several types of flavoprotein monooxygenases exist. One group catalyzes electrophilic aromatic substitution or heteroatom oxidation reactions, whereas the other group catalyzes Baeyer-Villiger-type oxidations of ketones (Fig. 2) (13, 17). 

Van Berkel WJ, Kamerbeek NM, Fraaije MW. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts review. J. Biotechnol. 2006 124 670-689. 

The mechanism for the hydroxylation of aromatic substrates by flavoprotein monooxygenases has been the subject of signiflcant research interest and controversy over the past decade. These enzymes (p-hydroxybenzoate hydroxylase, phenol hydroxylase, and melilotate hydroxylase) catalyze the initial step in the )8-ketoadipic acid pathway, the hydroxylation of substituted phenols into catechols (Scheme 55). Oxygen is required as cosubstrate, which is activated by the reduced FAD cofactor. The complex mechanism for the oxidative half-reaction is thought to consist of at least four steps and three intermediates 239-242) and to involve a controversial 4a,5-ring-opened flavin 242, 249, 250) (Scheme 56). The flavin C4a-hydroperoxy intermediate 64 and flavin C4a-hydroxy intermediate 65 have been assigned the structures shown in Scheme 56 based on the UV absorbance spectra of various model compounds compared with that of the modified enzyme cofactor alkylated at N(5) 243). However, evidence for the intermediacy of various ring-opened flavin species has been tentative at best, as model compounds and model reactions do not support such an intermediate 242). 

Scheme 55. General reaction scheme for the hydroxylation of aromatic substrates (S) by flavoprotein monooxygenases to give products (SO).

NADPH can serve as a cosubstrate of flavoprotein monooxygenase by first reducing the flavin, after which the reduced flavin can react with O2 to generate the hydroxylating reagent. An example is the bacterial 4-hydroxybenzoate hydroxylase which forms... 

A 4fl-hydroperoxy adduct (23) has been observed directly with flavoprotein monooxygenases [111,112]. These enzymes catalyze aromatic hydroxylation however their substrates are phenols rather than an unactivated phenyl ring. They do not... 

Similar behavior is observed with some of the flavoprotein monooxygenases, which also do not use a metal co-factor. The reduced flavin (68) has a structure which resembles that of luciferin (62) and reacts readily with molecular oxygen, through the intermediacy of its carbanion which forms charge-transfer intermediates leading to the hydroperoxide ion rather than to superoxide radical ion and pyrazyl radicals (158). Although the precise point of attachment of oxygen is controversial, the principle remains the same, namely the formation of a non-delocalized carbanion (69 or 70) (159—161). 

The flavoprotein monooxygenase or MFMO enzyme is indicated to be present in sponges (Kurelec et al. 1985, 1987). Post-mitochondrial supernatants of G. cydonium, V. aerophoba, P. semitubulosa and T. aurantium readily activated (metabolized) 2-aminoanthracene (AA) to S. typhinurium TA98 mutagens in an NADPH-dependent reaction. T. aurantium also activated aminobiphenyl, 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF). 

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