NewCat · Teaching Lytic Polysaccharide Monooxygenases to do Cytochrome P450 Catalysis

Lytic polysaccharide monooxygenases (LPMO) and cytochrome P450 (CYP) are copper- and iron-dependent, respectively,enzymatic systems that perform regio- and stereospecific oxidation of non-activated hydrocarbons in Nature. To control suchreactions in modern industry and biotechnology is of utmost importance in creating products of value such as secondgenerationbioethanol and products of value for i.e. the pharmaceutical industry. Due to the major drawbacks of using CYPs,including their partially membrane bound nature and the requirement of a reductase in combination with reducing agentssuch as NAD(P)H to transfer electrons to the active site for oxygen activation, it is highly desirable to develop new type ofcatalyst that can perform the same type of reactions. An attractive alternative strategy is to engineer LPMOs to perform CYPcatalysis. LPMOs are small, robust, easy to produce in large scale, and rigid water-soluble proteins with a plethora ofelectron donors. The extended, flat LPMO surface, with huge natural sequence variation and thus, likely, mutability, providesa fantastic scaffold for engineering access to the active site as well as substrate affinity. We propose to use LPMOsengineered to accommodate typical CYP substrates and immobilize this on solid supports to provide confinement necessaryin bringing the oxygen species together with the C-H bond to be oxidized in a tailored, ""closed"" environment. Moreover, therate of LPMO catalysis can be greatly enhanced compared to traditional CYP catalysis by the addition H2O2 in the presenceof low, priming concentrations of an external reductant to achieve efficiency constants (kcat/Km) in the order of 106 M-1s-1,which is typical for peroxygenases. The proposed ground-breaking research fits excellently well with the work program""Future and Emerging Technologies"" where the goal is to challenge current thinking.""