Professor Amanda Reig of Ursinus College

"Artificial Mini-Metalloenzymes for Hydrolytic Cleavage"

Metallohydrolases are metal-dependent enzymes that catalyze the hydrolytic cleavage of a range of biologically relevant substrates, including esters, organophosphates, polynucleotides, peptides, and CO2. The de novo designed Due Ferri single chain (DFsc) scaffold, a small computationally-designed four-helix bundle metalloprotein, is capable of stabilizing hydrolysis-prone metals such as Zn2+ and Ti4+ and hydrolyzing both small molecule substrates and double-stranded DNA. Importantly, the Ti4+-DFsc system is the first example of a functional titanium metalloenzyme and is an important step towards understanding the potential role for titanium in biological systems. To further understand the structural and electronic factors that contribute to the hydrolase reactivity in the DFsc system, a series of DFsc variants have been constructed and evaluated for their ability to bind metal ions and perform hydrolytic cleavage reactions using DNA gel electrophoresis, NMR, UV-visible absorbance, and fluorescence-based activity assays. The addition of an active site tyrosine residue, similar to that found in purple acid phosphatase, appears to confer increased hydrolytic activity.