Finding the Switch: Turning a Baeyer–Villiger Monooxygenase into a NADPH Oxidase

Abstract

By a targeted enzyme engineering approach, we were able to create an efficient NADPH oxidase from a monooxygenase. Intriguingly, replacement of only one specific single amino acid was sufficient for such a monooxygenase-to-oxidase switch-a complete transition in enzyme activity. Pre-steady-state kinetic analysis and elucidation of the crystal structure of the C65D PAMO mutant revealed that the mutation introduces small changes near the flavin cofactor, resulting in a rapid decay of the peroxyflavin intermediate. The engineered biocatalyst was shown to be a thermostable, solvent tolerant, and effective cofactor-regenerating biocatalyst. Therefore, it represents a valuable new biocatalytic tool.

Design of Artificial Alcohol Oxidases: Alcohol Dehydrogenase–NADPH Oxidase Fusions for Continuous Oxidations

“…During this reaction, the reactive peroxyflavin shunts back to the oxidized state, H 2 O 2 is formed, and NADP + is released. A mutant of a BVMO, phenylacetone monooxygenase (PAMO), was found to have a greatly enhanced uncoupling rate, thereby acting as an NADPH oxidase . Despite a profound change in activity, the C65D mutant PAMO was found to be as stable as the wild‐type PAMO, which is one of the most thermostable BVMOs characterized ( T m =60 °C).…”

Sweating the assets of flavin cofactors: new insight of chemical versatility from knowledge of structure and mechanism

“…The review period saw an elegant demonstration of such switching, namely conversion of a Baeyer-Villiger monooxygenase (BVMO) into a NADPH oxidase [27 ]. Most BVMOs can catalyze hydrogen peroxide formation in the absence of a suitable substrate but this so-called uncoupling reaction is typically very slow (<0.1 s À1 ).…”

Efficient Baeyer-Villiger electro-oxidation of ketones with molecular oxygen using an activated carbon fiber electrode in ionic liquid [bmim][OTf]

“…Various types of aryl, aliphatic, and naphthenic ketones can be successfully electro-oxidated to the corresponding esters and lactones with molecular oxygen (Table 3, entries [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], whereas the aryl ketones were less reactive, higher reaction constant current and longer reaction time were needed to reach good to excellent yields (Table 3, entries [1][2][3][4][5][6][7][8][9][10][11][12]. Various functionalities such as alkyl, alkoxy, bromo, trifluoromethyl, acetyl and nitro groups can tolerate the reaction.…”

“…Traditional methods for performing such a transformation generally involve the use of stoichiometric amount of the strongest oxidizing reagents (e.g., trifluoroperacetic acid, peroxyacetic acid, and perbenzoic acid) [5][6][7][8], and suffer from considerable drawbacks such as low yield, harsh or delicate reaction condition, and a large amount of waste by-products. Various reagents employed as the oxygen donors have been developed for this conversion include sodium perborate [9], potassium peroxydisulfate (K 2 S 2 O 8 ) [10,11], sodium perborate (NaBO 3 ) [11], m-CPBA [12,13], NADPH [14,15], phenylacetone monooxygenase (PAMO) [16], H 2 O 2 [17][18][19][20][21][22], oxone [23], h-SiO 2 ·KHSO 5 [24], and other complexes [25][26][27][28][29][30]. However, some of these procedures are invariably associated with one or more disadvantages such as the use of expensive reagents, long reaction time, difficulties in work up, environmental hazards, and difficulties in recycling of the catalyst.…”