Microbial Baeyer−Villiger Oxidation:  Stereopreference and Substrate Acceptance of Cyclohexanone Monooxygenase Mutants Prepared by Directed Evolution

Abstract

[reaction: see text] An array of random mutants of cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 was screened against a library of structurally diverse ketones for modifications in the substrate acceptance profile and stereopreference of the enzymatic Baeyer-Villiger biooxidation. While the set of mutant biocatalysts was initially evolved for the enantiocomplementary oxidation of 4-hydroxycyclohexanone, improved and/or divergent stereoselectivities were observed for several substrates. In addition, expanded substrate acceptance to facilitate biotransformation of sterically demanding ketones was found.

Baeyer−Villiger Monooxygenases: More Than Just Green Chemistry

“…264 Overall, the most interesting results were obtained with mutants containing amino acid substitutions at the 432 position as well as the double mutant (L426P/A541V). For example, two exo tricyclic ketones that were not substrates for the wild-type enzyme were recognized by two double mutants (L426P/A541V and F432Y/K500R) as well as the single mutant F432S.…”

Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

“…[34][35][36][37] Diese Enzyme sind in der Regel zu empfindlich, als dass sie in isolierter Form problemlos eingesetzt werden können. Leider wurde die Mehrzahl der organischen Chemiker nicht dazu ausgebildet, Tabelle 1: Oxidative Desymmetrisierung von Substraten unter Verwendung der CHMO-Mutante 1K2-F5 (Phe432Ser) mit Luftsauerstoff als Oxidationsmittel in einem Ganzzellenprozess.…”

“…Leider wurde die Mehrzahl der organischen Chemiker nicht dazu ausgebildet, Tabelle 1: Oxidative Desymmetrisierung von Substraten unter Verwendung der CHMO-Mutante 1K2-F5 (Phe432Ser) mit Luftsauerstoff als Oxidationsmittel in einem Ganzzellenprozess. [37] Substrat [39] Dieser Hinweis versetzte uns in die Lage, die stereochemischen Ergebnisse der CHMO-Mutante Phe432Ser zu deuten. Ein Homologiemodell der CHMO wurde mithilfe der Röntgen-struktur der PAMO konstruiert (Abbildung 2).…”

Enzymatic site-selectivity enabled by structure-guided directed evolution

“…65 Importantly, mutant F432S proved to be effective in the desymmetrization of a number of structurally different ketones without the need to perform additional mutagenesis experiments (Table 2). 66 In the kinetic resolution of a racemic ketone reported in an earlier study, epPCR was also applied, 63b although subsequently researchers turned away from ''blind'' directed evolution and opted for structure-guided generation of focused saturation mutagenesis libraries. 64 In spite of the impressive performance of CHMO variant F432S (Table 2), the system has limitations, including the insufficient robustness of this BVMO.…”

“…63a Blue arrow correlates with the (R)-enantiomer, red arrow with the (S)-antipode. Table 2 Oxidative desymmetrization of prochiral ketones using the CHMO mutant F432S in whole-cell reactions 66 Ser, and Gly). 67 This was the first case of using a reduced amino acid alphabet in directed evolution of stereoselective enzymes.…”