A recombination library of the best performing variants revealed substantially improved variants with triple and quadruple mutations. Variant L60F demonstrated a high chemoselectivity for the oxidation at the benzylic position, whereas L60F/S159G/A161F selectively formed aromatic ring oxidations with 2-methylnaphthalene. L60F also revealed an improved enantioselectivity up to 95 % ee for the conversion of indane to its benzylic hydroxylated derivative. MD simulations described a reshaping of MthUPO's active site during evolution due to the inclusion of bulkier residues, which induced novel binding modes for the studied substrates, and rationalised enzyme-substrate specific interactions providing the molecular basis for the experimentally observed oxidation patterns. These results demonstrate the high versatility and evolvability of MthUPO to achieve regio-, chemo-and enantioselective biocatalytic oxyfunctionalisations.