Directed evolution of decarboxylase OleT and nitrobindin hybrid catalysts
- Gelenkte Evolution der Decarboxylase OleT und von Nitrobindin-Hybridkatalysatoren
Markel, Ulrich; Schwaneberg, Ulrich (Thesis advisor); Okuda, Jun (Thesis advisor)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2021
Directed evolution is a powerful means to engineer enzymes specifically to our needs. In this thesis, the directed evolution of natural and artificial metalloenzymes (ArMs; also known as biohybrid catalysts) harboring porphyrin-based catalysts was successfully demonstrated. In the first part, a high-throughput screening (HTS) assay for decarboxylases was developed. So far, no HTS assay was available for OleT or other decarboxylases, which is why this important group of enzymes had no access to high-throughput protein engineering strategies yet. Using a diaryltetrazole as photoclickable dye, a robust 96-well photoclick assay for decarboxylases was developed. The assay does not rely on model substrates and detects styrene derivatives quantitatively in the micromolar range. In a directed evolution study the assay was applied to change the substrate specificity of the long chain fatty acid-preferring OleT-BM3R fusion enzyme toward the conversion of a non-natural aromatic substrate. Two enzyme variants with significantly changed substrate preference were identified (F79V and F79L). The presented photoclick chemistry-based HTS assay has potential implications for the directed evolution of OleT and other decarboxylases. In the second part of this thesis, ArMs containing porphyrin derivatives were successfully engineered. First the chemogenetic evolution of a peroxidase-like ArMs was demonstrated. In this regard, nitrobindin 4 (NB4) with its hydrophobic cavity design provided an ideal, evolutionary naïve starting point: It enabled the identification of the important distal R76 residue - a residue inaccessible when starting from wildtype nitrobindin due to the hydrogen bonding context within the wildtype’s cavity. Moreover, the open cavity of the NB4 scaffold allowed for peroxidase-like activity against a broad range of substrates using peracetic acid (AcOOH) as oxidant. Common redox mediators were converted with activity levels exceeding those of previous ArMs and rivaling those of natural peroxidases. Furthermore, three recalcitrant dyes were readily decolorized by the ArMs. Additionally, the combination of the MnPPIX cofactor and variants of the NB4 scaffold led to an artificial epoxygenase. Mechanistic investigations using UV/Vis spectroscopy indicate that the catalytic cycle proceeds through a Mn(V)=O and/or [Mn(IV)=O]<sup>+•</sup> species and show that the pH and the proximal histidine ligand play key roles in the activation of H<sub>2</sub>O<sub>2</sub>. The combination of the ArM with glucose oxidase in a cascade reaction ultimately allowed the direct utilization of molecular oxygen from air.
- RWTH PUBLICATIONS: RWTH-2021-08194