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

Abstract

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...

Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation

“…In the case of R292A, no product was detected with either 4-phenyl-2-butanone or 2-octanone, whereas the R292G enzyme showed a small residual activity of 1.5% and 4.5% with the same two ketones, respectively. As mentioned earlier, the arginine to alanine mutations in HAPMO and PAMO also resulted in inactive enzymes with no Baeyer-Villiger activity [37,38]. The observed lack of BaeyerVilliger activity of the two mutants towards the tested ketones confirms the importance of this arginine residue for catalysis [29].…”

“…For example, replacing R440 of HAPMO from Pseudomonas fluorescens by an alanine resulted in an inactive enzyme [37] while R337 of PAMO from Thermobifida fusca replaced by either alanine or lysine still formed the C4a-peroxyflavin intermediate but the enzyme was unable to perform either the Baeyer-Villiger reaction on phenylacetone or the S-oxidation on benzyl methyl sulfide [38]. More recently, a high-resolution crystal structure of PAMO from T. fusca [29] explained how the arginine 337 of this enzyme contributes to ketone activation via hydrogen bonding to the carbonyl oxygen of the substrate, thus increasing the electrophilicity of the carbonyl carbon to enhance the nucleophilic attack by the C4a-peroxyflavin intermediate.…”

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ 3 -4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

“…The corresponding R440A mutation in 4-hydroxy-acetophenone monooxygenase (HAPMO) results in an inactive BVMO, although the reduction of the flavin by NADPH is not affected (28,60). Similarly, the R337A and R337K PAMO mutant enzymes competently form the C-4a peroxyflavin intermediate but cannot effect catalysis with substrates, indicating a key catalytic role (57).…”

Investigation of the ligand spectrum of human sterol carrier protein 2 using a direct mass spectrometry assay

“…This coil-helix transition upon ligand binding may to some degree explain the ''tightening'' of secondary structure we observe when SCP2 is loaded with stearoyl CoA. ''Soft'' ESI-MS has frequently been used to study biomolecular complexes in a nearly native state in the gas phase, for example protein/protein [35], enzyme/cofactor [36] and protein/lipid [25,37]. We have adapted this kind of assay to analyse SCP2/lipid.…”