Friday, September 17, 2021
Host: Dr. Aaron Smith
Developing hydrolytic mini-metalloenzymes using de novo proteins
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 nerve gases). While metal ions are not required for hydrolytic cleavage reactions, they can be particularly powerful catalysts due to their high Lewis acidity, resulting in a metal-bound hydroxide at neutral pH. When placed within a protein scaffold, the resultant highly reactive hydroxide ion can be positioned for optimal nucleophilic attack on a substrate, allowing for hydrolysis under room temperature and neutral pH conditions. 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 Å 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 further modified this site to include a tyrosine, which is found in the active site of purple acid phosphatase, and known to bind and stabilize hydrolysis-prone metals. We have tested the effect of this substitution on both metal binding and hydrolytic activity, making mini-metalloenzymes (MMEs). We have studied metals commonly used by nature, like Zn2+, as well as metals underutilized by nature, like Ti4+ and V5+, for their ability to bind to these protein scaffolds, and have seen differential binding in constructs containing this tyrosine substitution vs. those that do not. We have also tested the effect of both metal and protein sequence on their ability to hydrolytically cleave DNA and have seen both successful cleavage and increased activity with the tyrosine substituted proteins.