FAD-4a-OOH

All mechanistic studies on enzymatic Baeyer-Villiger reactions support the hypothesis that conventional and enzymatic reactions are closely related [1063, 1204]. The oxidized flavin cofactor (FAD-4a-OOH, see Scheme 2.147) plays the role of a nucleophile similar to the peracid. The strength of enzyme-catalyzed Baeyer-Villiger reactions resides in the recognition of chirality [1205-1207], which has been accomplished by conventional means only recently, albeit in reactions exhibiting moderate selectivities [1208]. 

The CHMO from Acinetobacter sp. NCIMB 9871 has been studied quite extensively. The enzyme is a monomer (M, 66,000) contains a noncovalently bound FAD molecule and uses NADPH as the electron donor. This enzyme was cloned and expressed in E. coli [18]. The mechanism of action of CHMO was proposed by Walsh et al. [19] (Fig. 6). It proposes that the enzyme-FAD-4a-OOH is the nucleophilic peroxide equivalent that produces the tetrahedral adduct with bound substrate. The adduct then decomposes by the migration of carbon-carbon bond to the proximal peroxide oxygen as the 0-0 bond breaks. This produces the ring expansion to lactone and the enzyme-FAD-4a-OH spontaneously eliminates H2O to regenerate the oxidized FAD. 

However, recent x-ray studies on p-hydroxybenzoate-p-hydroxybenzoate hydroxylase binary complex crystals clearly show the aromatic substrate is bound at the flavin 4a-5 edge and orthogonal to the isoalloxazine plane (29). Unless this binary complex structure is highly misinformative, it can be inferred that in the 02, p-hydroxy-benzoate, enzyme ternary active complex, oxygen transfer is in the 4a,5 region, not the la, 1 region of the bound FAD, which rules out la-OOH derivatives as important oxygenating intermediates for this enzyme. 

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