Crystal structure of a Baeyer–Villiger monooxygenase

Abstract

Flavin-containing Baeyer-Villiger monooxygenases employ NADPH and molecular oxygen to catalyze the insertion of an oxygen atom into a carbon-carbon bond of a carbonylic substrate. These enzymes can potentially be exploited in a variety of biocatalytic applications given the wide use of Baeyer-Villiger reactions in synthetic organic chemistry. The catalytic activity of these enzymes involves the formation of two crucial intermediates: a flavin peroxide generated by the reaction of the reduced flavin with molecular oxygen and the ''Criegee'' intermediate resulting from the attack of the flavin peroxide onto the substrate that is being oxygenated. The crystal structure of phenylacetone monooxygenase, a Baeyer-Villiger monooxygenase from the thermophilic bacterium Thermobifida fusca, exhibits a two-domain architecture resembling that of the disulfide oxidoreductases. The active site is located in a cleft at the domain interface. An arginine residue lays above the flavin ring in a position suited to stabilize the negatively charged flavin-peroxide and Criegee intermediates. This amino acid residue is predicted to exist in two positions; the ''IN'' position found in the crystal structure and an ''OUT'' position that allows NADPH to approach the flavin to reduce the cofactor. Domain rotations are proposed to bring about the conformational changes involved in catalysis. The structural studies highlight the functional complexity of this class of flavoenzymes, which coordinate the binding of three substrates (molecular oxygen, NADPH, and phenylacetone) in proximity of the flavin cofactor with formation of two distinct catalytic intermediates.flavoenzyme ͉ mechanism of catalysis ͉ biocatalysis ͉ Baeyer-Villiger reaction ͉ crystallography A t the end of the 19th century, Baeyer and Villiger (1) discovered that cyclic ketones react with oxidants, such as peroxymonosulfuric acid, to yield lactones. The mechanism of the Baeyer-Villiger reaction involves a nucleophilic attack of a peroxide to the ketone reagent to generate the so-called ''Criegee'' intermediate ( Fig. 1), which is followed by an intramolecular rearrangement that leads to the migration of an alkyl group to an oxygen atom, generating the lactone product. Baeyer-Villiger reactions are of enormous value in synthetic organic chemistry, and the number of their applications is countless.Several microorganisms produce enzymes capable to catalyze Baeyer-Villiger reactions. These proteins are extensively studied for their exploitation in biocatalytic applications (2, 3). This interest follows the problems related to the toxicity and instability of the oxidizing reactants that are currently being used in chemical processes. In addition, enzymatic reactions exhibit a superior degree of enantio-and regioselectivity. Baeyer-Villiger monooxygenases are classified depending on the nature of their flavin cofactor (4). The type I enzymes are the most extensively investigated (5). They are FAD-dependent proteins that use NADPH and molecular oxygen to insert an oxygen atom into th...

Mechanism of action of a flavin-containing monooxygenase

“…This difference may explain why NADPH was not observed in the structure of BVMO when cocrystallized with NADPH (16). Structural differences in the substrate-binding channel almost certainly explain the difference in substrate selectivity of these enzymes.…”

“…FMO has two well defined structural domains, whereas the others have three. The BVMO structure has two structural domains within the insertion in addition to the large domain (16). Other oxidoreductases, such as NADH peroxidase and lipoamide dehydrogenase, have one small insertion domain and two domains, whereas FMO and BVMO have only one domain (17,18).…”

Structure of the monooxygenase component of a two-component flavoprotein monooxygenase

“…An accurate comparison between C 2 and these structural homologs reveals that the main differences are restricted to the loops lining the flavin-and substrate-binding sites. The folding topology of C 2 is unrelated to that exhibited by other flavin-dependent monooxygenases/hydroxylases of known three-dimensional structure, such as p-hydroxybenzoate hydroxylase (15), yeast flavin-containing monooxygenase (16), and phenylacetone monooxygenase (17).…”

Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

“…Because structural features that are unique to FMO, and yet define FMO as a family, are least amenable to modeling a priori, crystallization of an FMO remains essential to gaining an in-depth understanding of the structure and function of this family. The only bacterial flavin monooxygenase whose crystal structure has been solved is phenylacetone monooxygenase (PAMO; Malito et al, 2004). The authors found significant homology with human GR (ca.…”