***PLEASE NOTE: ongoing NAISS 2025/22-795 is for an international PhD-school*** This project is funded by VR and Novo Nordisk Foundation individual grants to Per-Olof Syrén as well as by SSF, MISTRA, FORMAS. Published papers using NAISS 2024/5-346 4, and 1 manuscript to be submitted. This multicenter project will use de novo enzyme design to bridge transition metal catalysis with protein chemistries for the generation of new-to-nature enzymatic reactions. Enzymes show a great potential towards reaching a more sustainable society by allowing for specific transformations starting even from complex biomass under mild conditions. However, for many industrially important chemical reactions, an enzyme is lacking. This is particularly true for carbon-carbon and carbon-heteroatom bond formation which is of basal importance to make platform chemicals, materials, agrochemicals and pharmaceuticals. We are thus still today often limited to harsh chemical synthesis relying on petroleum-based feedstock and hazardous reagents and conditions. Here, we will create metalloenzymes de novo to create carbon-carbon bond forming enzymes with broad catalytic scope. Using computational methods spanning from quantum mechanics (QM), molecular dynamics (MD) and generative AI-based protein design we will fill current gaps in available biocatalytic transformations. We are focusing on cross-coupling reactions for which our devised pipeline of protein design consists of: 1) QM-calculation of a transition state for the relevant reaction (here cross-couplings) 2) Generative AI to diffuse a protein backbone capable of harboring this reaction, accounting for intermediates and necessary catalytic amino acids 3) Validation by AlphaFold followed by MD and docking to find hot-spots amenable for further engineering. Designs are tested in the lab experimentally and further engineered by directed evolution to reach efficient catalytic rates. By implementing new reactions and mechanisms in de novo metalloenzymes guided by high-level QM calculations and experimental data, the result are designer enzymes capable of harnessing chemical transformations of high industrial and societal relevance that are currently not accessible by biocatalysis. The PI has experience in modelling reaction mechanism in enzymes and great progress was made in previous PDC-supported projects, as we unraveled new chemistries in existing enzymes. For example, we could develop a new set of catalysts for plastic degradation using enzyme catalysis. This demonstrates necessary expertise of the main applicant in performing state of the art enzyme design (see e.g. Syrén et al. J. Org. Chem. 2018, Nat. Commun. 2023, Green. Chem. 2024). Motivation to Multicenter usage: Dardel: Per-Olof Syrén (PI) has several funded projects from VR, FORMAS, MISTRA and SSF whose success are entirely dependent on running US-GAMESS. Calculations are based on very large models of complex biocatalysts containing metals. Tetralith: This project is dependent on GPUs to run molecular dynamics (openMM) and generative AI for protein structure prediction (AlphaFold) and design. We also use Schrödinger for docking. Ca 6 people from my group will use these resources