Flavoprotein monooxygenase mechanism

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

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). 

Mechanism of the enzymatic sulfoxidation of prochiral sulfides catalyzed by flavoprotein monooxygenases. 

Suske, W. A., W. J. H. van Berkel, and H.-P. E. Kohler, Catalytic mechanism of 2-hydroxybiphenyl 3-monooxygenase, a flavoprotein from Pseudomonas azelaica HBP1 , J. Biol. Chem., 247, 33355-33365 (1999). 

Studies with cell-free hydroxylases suggest that the hydroxylation mechanisms are complex. It is assumed that an electron transport system involving an NADPH-dependent flavoprotein, an iron-sulfur protein, and cytochrome P-450 is involved. In the case of the steroid 15/S-hydroxylase system of Bacillus megaierium, these three components have been demonstrated15. The 1 la-hydroxylase of Rhizopus nigricans is also an enzyme of the P-450 monooxygenase type which works with an NADPH-cytochrome P-450 reductase. In this case the enzyme complex is associated with the endoplasmic reticulum of the mycelial cells34. 

The catalytic cycle of each flavoenzyme consists of two distinct processes, the acceptance of redox equivalents from a substrate and the transfer of these equivalents to an acceptor. Accordingly, the catalyzed reactions consist of two half-reactions a reductive half-reaction in which the flavin is reduced and an oxidative half-reaction, in which the reduced flavin is reoxidized. This review summarizes the chemistry of simple flavoprotein reductases, dehydrogenases, (di)thiol oxidoreductases, oxidases, and monooxygenases (Table 1) (5 0) This grouping provides a good appreciation about what type of common mechanisms can be distinguished and what type of substrates can be converted. Information on the chemistry of complex flavoenzymes can be found in the Further Reading section. 

To clarify the reaction mechanism of monooxygenase, we have recently purified two monooxygenases from pseudomonads. Imidazoleacetate monooxygenase (5) and L-lysine monooxygenase (JO) were obtained in crystalline form, and both were shown to be flavoproteins. The former requires an exogenous hydrogen donor, but the latter utilizes the hydrogen atoms of L-lysine. 

Insertion of the C5,6 double bond (A -> A ) involves removal of the 5a (ex 4-pro-R H of MVA) and 6a (ex 5-pro-S H of MVA) hydrogens. In rat liver and yeast microsomal preparations the reaction requires aerobic conditions and NADH or NADPH and both of the eliminated hydrogens are found in HjO. This suggests a mechanism in which hydroxyla-tion at CS or C6 is followed by dehydration across these two carbons or, alternatively, a fatty acid-type desaturation. There is evidence of the former in yeast, whereas the latter seems more likely in rat liver where the multienzyme complex catalysing it consists of the A -desaturase itself, cytochrome and an NAD(P)H-dependent flavoprotein the A -desaturase is a monooxygenase whieh uses electrons derived from NAD(P)H, via the flavoprotein and cytochrome i>5, and O2 to effect the removal of the two hydrogens from the sterol. 

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