Engineering de novo metal-protein complexes as artificial enzymes for carbon-carbon bond formation

• Applicant: Professorin Dr. Cathleen Zeymer
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 453748800

The chemical and pharmaceutical industry is under increasing pressure to replace traditional chemical catalysis with environmentally benign approaches for the synthesis of high-value compounds. Using enzymes as catalysts promises sustainability in combination with superior selectivity and catalytic efficiency. In many cases, however, natural enzymes do not promote the abiological reactions relevant for industrial processes. A powerful strategy to broaden the scope of biocatalysis is the development of artificial metalloenzymes, in which a catalytically active metal cofactor is embedded in a protein scaffold that provides stereoselectivity. My group has recently established the formation of robust and specific metal-protein complexes based on computationally designed protein scaffolds and non-natural metal cofactors. Notably, the metals are anchored via direct coordination by natural amino acids of the protein. Building on this work, we now aim to engineer these de novo complexes in order to generate a versatile biocatalytic platform for synthetically valuable reactions, including stereoselective carbon-carbon bond formations. The first part of the proposal focuses on an artificial di-rhodium enzyme to catalyze carbene insertion reactions, while the second part introduces a lanthanide enzyme catalyzing Michael additions. Our approach for catalyst optimization exploits directed evolution, a technique that mimics natural evolution in the laboratory through iterative cycles of mutagenesis and in vitro screening. Improvements in product yield, total turnover number, regio-/stereoselectivity, and catalytic efficiency can typically be achieved due to subtle structural rearrangements induced by a small number of beneficial mutations. However, fully computationally designed proteins have not yet been subjected to extensive laboratory evolution and we thus aim to assess their evolvability. Furthermore, structural and mechanistic characterization of the engineered enzymes is an integral part of the work and essential to understand the mechanism of action. If successful, this work will not only lead to innovative biocatalytic applications, but will also provide valuable mechanistic insights that can be transferred to both synthetic and natural bioinorganic catalytic systems. Moreover, we aim to contribute to a fundamental understanding of how to generate an enzyme from scratch.

• DFG Programme: Research Grants