Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase.
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.
van den Heuvel
Robert H H
RH
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University. r.h.h.vandenheuvel@chem.uu.nl
Tahallah
Nora
N
Kamerbeek
Nanne M
NM
Fraaije
Marco W
MW
van Berkel
Willem J H
WJ
Janssen
Dick B
DB
Heck
Albert J R
AJ
D002384:Catalysis; D003001:Cloning, Molecular; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D006358:Hot Temperature; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D009249:NADP / Q000031:analogs & derivatives / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011551:Pseudomonas fluorescens / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors
D007659:Ketones; D009249:NADP; C024375:3-aminopyridine adenine dinucleotide phosphate; D010105:Oxygenases; C427385:4-hydroxyacetophenone monooxygenase; D010100:Oxygen