4‐Hydroxyacetophenone monooxygenase from <i>Pseudomonas fluorescens</i> ACB

Kamerbeek, Nanne M.; Moonen, M.J.H.; Ven, Jos G.M. van der; Berkel, Willem J.H. van; Fraaije, Marco W.; Janssen, Dick B.

doi:10.1046/j.1432-1327.2001.02137.x

Cited by 135 publications

(86 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3 and 6) is present in this position, which, therefore, appears to be able to bind a negatively charged group. In (36). The sequence alignment was calculated with CLUSTALW (37).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of a Baeyer–Villiger monooxygenase

Malito

Alfieri²,

Fraaije³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

275

305

View full text Add to dashboard Cite

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...

show abstract

“…3 and 6) is present in this position, which, therefore, appears to be able to bind a negatively charged group. In (36). The sequence alignment was calculated with CLUSTALW (37).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of a Baeyer–Villiger monooxygenase

Malito

Alfieri²,

Fraaije³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

275

305

View full text Add to dashboard Cite

show abstract

“…1), thereby acting as an NADPH oxidase. The observed oxidase activity of HAPMO in the absence of an aromatic substrate is 0.1 s Ϫ1 (13), which may suggest that also with HAPMO, NADP ϩ remains bound throughout the oxidative half-reaction. Kamerbeek et al (21) do not address the issue of protein-coenzyme complexes present during the catalytic cycle.…”

mentioning

confidence: 89%

“…4 HAPMO from Pseudomonas fluorescens ACB is a Baeyer-Villiger monooxygenase (BVMO), which converts a wide range of ketones into the corresponding esters or lactones (13). The enzyme is also able to perform enantioselective sulfoxidations (14).…”

mentioning

confidence: 99%

Coenzyme Binding during Catalysis Is Beneficial for the Stability of 4-Hydroxyacetophenone Monooxygenase

Heuvel¹,

Tahallah²,

Kamerbeek

et al. 2005

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP ؉ only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP ؉ interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP ؉ ) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.The interaction of proteins with small molecules, such as ligands and cofactors, often coincides with an increased stability of the protein due to the coupling of binding with the unfolding equilibrium (1-4). Thus, apart from their catalytic role, cofactors may also have a structural role. The importance of cofactor binding for protein stability is revealed for several flavoproteins (2). The common polymorphism 677TC 3 T in methylenetetrahydrofolate reductase causing the single point mutation A222V reduces the affinity of the enzyme for the FAD cofactor, resulting in a lower thermal stability (5, 6). For the octameric protein vanillylalcohol oxidase, it was demonstrated that cofactor binding influences the quaternary architecture of the enzyme (7). The H61T mutant is purified as apo-enzyme and mainly exists as a dimeric species. The binding of FAD to the enzyme restores the octameric conformation (7,8). Similarly, for lipoamide dehydrogenase, it was shown that FAD binding increases the protein melting temperature from 35 to 80°C (9). Coenzymes such as NAD(P) ϩ and NAD(P)H can have, besides from their function in electron transfer, also a structural role. Several liver and pectoral muscle enzymes are substantially protected by NAD(P) ϩ against heat and proteolysis by trypsin (10). Phosphite dehydrogenase is stabilized by NAD ϩ (11), and DNA ligases become more compact upon anchoring coenzyme NAD ϩ (12).In the present work, we have studied the noncovalent interactions between the oxidized...

show abstract

“…4) The latter is converted to 4-hydroxyphenyl acetate by 4-hydroxyacetophenone monooxygenase (EC 1.14.13.84), and subsequently is hydrolyzed to hydroquinone. [8][9][10] Hydroquinone is cleaved to 4-hydroxymuconic semialdehyde and metabolized further. 11) Besides these metabolic intermediates, a small amount of 2,4 0 -dihydroxyacetophenone (DHAP, 2-hydroxy-1-(4-hydroxyphenyl)ethanone) is formed as a dead-end product, which is not metabolized any further but accumulates in the culture broth of bisphenol A-assimilating bacterium.…”

mentioning

confidence: 99%