Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis

Abstract

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

Fig. 1.

Active-site residues of PAMO targeted in the mutagenesis study. FAD is shown in black. For clarity, only the side chains of amino acid residues are presented. Proposed mutations are indicated. The schematic was prepared using PyMol software and the structure of PAMO (PDB ID 1W4X_A).

Fig. 2.

Sequence alignment of PAMO and CPMO. Mutations proposed after the initial analysis are indicated with an asterisk (*), while mutations proposed after the analysis of the new structure of PAMO are indicated with a pound sign (#).

Fig. 3.

Substrates used in the whole-cell screening (A) and in conversions with the isolated enzymes (B).

Fig. 4.

(A) The Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one (racemic compound 2) leads to the formation of the “normal” (2a) and “abnormal” (2b) lactones. (B) Similarly, 1-indanone (4) can be oxidized to the “normal” (4a) and “abnormal” (4b) products.

Fig. 5.

A schematic representation of the active site of PAMO. Residues identified as hot spots for substrate specificity are displayed as sticks. Residue R337 that is essential for catalysis is also shown. FAD is presented as black sticks. The schematic was prepared using PyMol software and the structure of PAMO (PDB ID 1W4X_A).