Engineering the SpyTag peptide for artificial stereoselective metalloenzymes: copper coordination and catalytic function

"Metalloproteins facilitate some of the most intricate biological processes in nature. The project described here involves the redesign of the Spy complex, which comprises a peptide (SpyTag) that forms an isopeptide bond with a protein (SpyCatcher), resulting in a recombined Spy protein (Figure 1a). The peptide component allows for the straightforward incorporation of metal-binding sites into the final protein without the need to redesign the entire structure. Additionally, peptides can be synthesized using solid-phase methods, enabling the inclusion of non-natural amino acids. This expands the potential applications of the Spy protein in fields such as industry, biotechnology, and nanotechnology." We studied second generation of SypTag peptides bearing bis-histidine sites, capable to bind Cu(II) and to promote reactions of oxidation of catechols. The affinity constants of the SpyTag peptides for Cu(I) and Cu(II) were determined by spectrophotometric and spectrofluorimetric titrations. Also, an evaluation of the kinetics in the oxidation of catechol, like 4-methylcatechol (figura 1b), in the presence of the Cu(II)/peptide adducts was performed. After that, DOPA in its two enantiomers was used to evaluate possible stereoselectivity in substrate oxidation.