Active Site Engineering of Three Scaffolds for Artificial Metalloenzyme-Assembly and Applications Thereof

This PhD thesis summarizes the work carried out in the research group of Prof. Dr. Ward at the University of Basel during the years of 2018 – 2022. The Ward group utilizes several scaffolds for the development of novel artificial metalloenzymes with functions spanning over ring-closing metathesis, C-H activation, deallylation, transfer
hydrogenation, Suzuki-coupling and more. Artificial metalloenzymes are hybrid catalysts that combine attractive features of both enzyme and homogeneous catalysis. They are assembled by the incorporation of catalytically active metal-containing moieties into protein scaffolds.
There are two options to alter the performance of the artificial metalloenzyme: i) by chemical modification of the metal cofactor or ii) by modification of the second coordination sphere which is formed by the protein scaffold as a defined reaction environment around the metal cofactor. This work focused on the latter by the following strategies: i) (rational) protein design, ii) high-throughput screening, iii) unnatural amino acid incorporation and iv) directed evolution. An additional project dealt with the in vivo uncaging of a drug in the tumor microenvironment of hypoxic carbonic anhydrase IX expressing tumor cells.
(Rational) Protein Design: Human carbonic anhydrase II was used to exploit a dual anchoring strategy to improve the localization of an arylsulfonamide-bearing iridiumpianostool
catalyst within the scaffold to yield artificial metalloenzymes with increased activity and enantioselectivity in transfer hydrogenation. Furthermore, both streptavidin and human carbonic anhydrase II were utilised in computational design campaigns to modify the artificial metalloenzymes’ catalytic sites by domain grafting into a selected loop of the respective scaffold. 135 streptavidin variants and 16 human carbonic anhydrase II variants were selected as a computationallyguided
library. The streptavidin variants were subsequently subjected to functional and biochemical characterization.
Directed Evolution: In a third project, HaloTag was subjected to directed evolution to improve its maximum fluorogenicity and the apparent second-order bioconjugation rate constants in combination with a styrylpyridium dye. Crystallographic characterization of selected mutants carried out in the course of this thesis revealed the chemical origin of the fluorescent enhancement.
High-throughput Screening: A site-saturation library at positions S112 and K121 within the streptavidin scaffold comprising of mutants with all 400 possible combinations was prepared by golden-gate cloning and Gibson assembly. This library was used for high-throughput screening to find artificial metalloenzymes based on a copper tris(azolyl)borate that enable C(sp3)-H functionalization via intramolecular carbene insertion.
Unnatural Amino Acid Incorporation: Several trials were performed to introduce unnatural amino acids into streptavidin. However, introduction into this scaffold proved challenging as described in Appendix A. Therefore, HaloTag was selected as a host for the introduction of unnatural amino acids. Four metal-chelating unnatural amino acids were incorporated into HaloTag in three selected sites. Variants featuring a hydroxyquinoline-bearing side chain in combination with [(η5-C5H5)Ru(MeCN)3]PF6 generated artificial metalloenzymes that can perform allylic deamination reactions.
In Vivo Uncaging of an Antineoplastic Agent in HeLa Cell Cultures: HeLa cells were subjected to hypoxia to induce high expression of carbonic anhydrase IX on cell surfaces. Subsequently, a ruthenium containing cofactor with a sulfonamide anchor was incubated with the cells and washed off before inducing catalysis by adding caged monomethyl auristatin E.
In summary, these efforts introduce various new functions into three protein scaffolds for artificial metalloenzyme-assembly and provide the groundwork for using shielded active sites in streptavidin or human carbonic anhydrase II for different reactions currently pursued in the group.