Self‐Sufficient Baeyer–Villiger Monooxygenases: Effective Coenzyme Regeneration for Biooxygenation by Fusion Engineering

Abstract

Two‐in‐one biocatalysts were engineered by the covalent fusion of NADPH‐dependent Baeyer–Villiger monooxygenases to a phosphite dehydrogenase for coenzyme regeneration (see scheme). Not only the purified fusion proteins, but also whole cells and crude cell extracts containing the enzyme conjugates, could be used to catalyze biotransformations with high efficiency. NADP+=nicotinamide adenine dinucleotide phosphate.

Snapshots of Enzymatic Baeyer-Villiger Catalysis

“…The three-dimensional structure of PAMO has been solved revealing a two-domain organization (Fig. 1A) (16), and its catalytic cycle has been subject of extensive kinetic studies (17)(18)(19)(20)(21)(22). More recently, the structure of cyclohexanone monooxygenase in complex with NADP ϩ highlighted a proposed mechanism for the dual catalytic role of NADP(H) (9,23).…”

Aerobic Double Dehydrogenative Cross Coupling between Cyclic Saturated Ketones and Simple Arenes

“…Inspired by these cited examples, and the previous reports of successful Type I BVMO-based fusion proteins (Torres Pazmiño et al, 2008, 2998, attempts were made in this study to create functional fusions of Fre and the oxygenating component of either 2,5-or 3,6-DKCMO within one single protein using two different linkers and two different relative orientations of the FR and 2,5-or 3,6-DKCMO oxygenating component genes to generate several different organisational variants (Table 2). While the FR activity of all seven investigated fusion proteins was not influenced significantly by being concatenated with either of the larger oxygenating subunits, all of the tested combinations exhibited a substantial detrimental effect on the activity of the monooxygenase protein moiety, and considerable decomposition was observed in all cases.…”

“…The generation of Type II BVMO-based fusion proteins was considered to be particularly challenging, because although catalytically active fusion proteins created out of various Type I BVMOs together with phosphite dehydrogenase to achieve self-sufficient enzymes with respect to recycling the requisite nicotinamide cofactor have been reported (Torres Pazmiño et al 2008, the relative complexity of successfully replacing the evolved substrate shuttle role of FMN in Type II BVMOs was likely to be more difficult to achieve.…”

Immobilized redox enzymatic catalysts: Baeyer–Villiger monooxygenases supported on polyphosphazenes

“…This novel biocatalyst would resemble a 'self-sufficient' redox enzyme [23,24], where the BVMO and the dehydrogenase are covalently linked.…”

“…One of the main drawbacks for scaling-up BVMOcatalyzed reactions is the need of the expensive nicotinamide cofactor NAD(P)H. For this, usually another enzyme like glucose-6-phosphate dehydrogenase (G6PDH) is employed coupled with the BVMO to allow the cost effective utilization of a catalytic amount of the cofactor. Recently, a more sophisticated 'self-sufficient' approach was described by covalently fusing both BVMO and a recycling enzyme [23,24]. In this case, several BVMOs were linked to a phosphite dehydrogenase (PTDH), which catalyzes the oxidation of phosphite into phosphate by which it reduces NADP + into NADPH.…”