Teaching Lytic Polysaccharide Monooxygenases to do Cytochrome P450 Catalysis

Introduction

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.

Importance in Industry

To control such reactions in modern industry and biotechnology is of utmost importance in creating products of value such as second-generation bioethanol and products of value for, e.g., the pharmaceutical industry.

Drawbacks of CYPs

Due to the major drawbacks of using CYPs, including:

• Their partially membrane-bound nature
• The requirement of a reductase in combination with reducing agents such as NAD(P)H to transfer electrons to the active site for oxygen activation

it is highly desirable to develop a new type of catalyst that can perform the same type of reactions.

Alternative Strategy

An attractive alternative strategy is to engineer LPMOs to perform CYP catalysis. LPMOs are:

• Small
• Robust
• Easy to produce in large scale
• Rigid water-soluble proteins with a plethora of electron donors

The extended, flat LPMO surface, with huge natural sequence variation and thus, likely, mutability, provides a fantastic scaffold for engineering access to the active site as well as substrate affinity.

Proposed Methodology

We propose to use LPMOs engineered to accommodate typical CYP substrates and immobilize this on solid supports to provide confinement necessary in bringing the oxygen species together with the C-H bond to be oxidized in a tailored, "closed" environment.

Enhanced Catalysis

Moreover, the rate of LPMO catalysis can be greatly enhanced compared to traditional CYP catalysis by the addition of H2O2 in the presence of low, priming concentrations of an external reductant to achieve efficiency constants (kcat/Km) in the order of 10^6 M^-1s^-1, which is typical for peroxygenases.

Alignment with Research Goals

The proposed ground-breaking research fits excellently well with the work program "Future and Emerging Technologies" where the goal is to challenge current thinking.