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

Marco W. Fraaije,Dick B. Janssen,Nanne M. Kamerbeek,Nora Tahallah,Albert J.R. Heck,Robert H.H. van den Heuvel,Willem J.H. van Berkel

doi:10.1074/jbc.m503758200

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.

Highlights

The quaternary architecture of the enzyme [7]
Quaternary Structure of hydroxyacetophenone monooxygenase (HAPMO) Variants—Wild type HAPMO is a homo-dimeric flavoprotein with each monomer containing a noncovalently tightly bound FAD cofactor [13]
We have reported the first direct analysis of the noncovalent interactions of a flavin-containing Baeyer-Villiger monooxygenase with its nicotinamide coenzyme during the enzyme catalytic cycle

Summary

Introduction

The quaternary architecture of the enzyme [7]. The H61T mutant is purified as apo-enzyme and mainly exists as a dimeric species. This first structure of a type I BVMO revealed a two-domain architecture and highlighted the importance of an active site arginine, which seems critically involved in substrate oxygenation Replacement of this conserved arginine in HAPMO (R440A) does not impair NADPH binding but results in complete enzyme inactivation [21]. A similar kinetic mechanism has been proposed for pig liver flavin-containing monooxygenase [23], suggesting that continued association of coenzyme is a recurrent theme for this class of monooxygenases, regulating enzyme activity It has been found for these monooxygenases that when no suitable substrate is present, the flavin C4a-peroxide will slowly decay to form hydrogen peroxide while releasing NADPϩ (Fig. 1), thereby acting as an NADPH oxidase. The data in this report have provided the first direct evidence that the nicotinamide coenzyme, either in its reduced or in its oxidized form, interacts with HAPMO throughout the catalytic cycle and stabilizes the enzyme to a great extent

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Results

Conclusion