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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 342487
Title Coenzyme binding is beneficial for the stability of 4 hydroxyacetophenone monooxygenase
Author(s) Heuvel, R.H.H. van den; Tahallah, N.; Kamerbeek, N.M.; Fraaije, M.W.; Berkel, W.J.H. van; Janssen, D.B.; Heck, A.
Source Journal of Biological Chemistry 280 (2005)37. - ISSN 0021-9258 - p. 32115 - 32121.
Department(s) Biochemistry
Publication type Refereed Article in a scientific journal
Publication year 2005
Keyword(s) baeyer-villiger monooxygenases - ionization mass-spectrometry - vanillyl-alcohol oxidase - electrospray-ionization - escherichia-coli - methylenetetrahydrofolate reductase - cyclohexanone monooxygenase - synthetic applications - protein complexes - specificity
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.
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