Abstract 2339 Hydrolytic mini-metalloenzymes

bioinorganic chemistry, de novo protein design, structure function relationships Metallohydrolases are metal-dependent enzymes that catalyze the hydrolytic cleavage of a range of biologically relevant substrates, including esters, organophosphates, polynucleotides, peptides, and CO2.They are found throughout all kingdoms of life and their biological functions make them valuable potential targets for drug design (e.g., osteoporosis, cancer, cystic fibrosis), antibacterials, and bioremediation catalysts (e.g., degradation of pesticides or organophosphorus nerve gases).Metallohydrolases can be mononuclear or multinuclear, and function with a wide range of metal ions, including Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Cu2+, Ni2+, and Zn2+.Binuclear metallohydrolases contain two metal ions spaced 3-4 apart coordinated primarily by a combination of Asp, Glu, and His amino acids.Utilizing the de novo designed Due Ferri single chain (DFsc) scaffold, we have made a series of proteins, which feature these residues in a binuclear active site, and have identified a pair of proteins where one is completely unfolded without metal and completely folded with metal, and a second is folded regardless of metal binding.We have tested the effect of this difference on both metal binding and hydrolytic activity with our mini-metalloenzymes (MME).We have assessed binding via circular dichroism and inductively coupled plasmaoptical emission spectroscopies, and hydrolytic activity for a range of natural (Zn, Mn, Fe, Co) metal ions by using small molecule colorimetric assays and monitoring DNA cleavage activity using gel electrophoresis.We have seen that reactivity is well correlated to metal binding, and not just protein structure, but does not have a direct correlation to metal binding affinity of the proteins.These systematic studies provide the basis for understanding the structural factors that influence metal preference and substrate reactivity in natural binuclear metallohydrolases.