Our first proof of principle study focuses on the enzymatic process of acetylation.
Acetylation is a common but poorly understood co-translational and post-translational protein modification in which an acetyl group is attached to protein residues. Acetyltransferases are responsible for this process in cells and require the presence of the cofactor Acetyl Coenzyme-A. The acetyltransferase enzyme family has specific members that can catalyze the transfer of an acetyl group to a lysine side chain (KAT) or the N-terminus (NAT) of a protein. In my lab, we identify amino acids on acetyltransferases where we hypothesized that changes in specific structural features might contribute to substrate specificity. We do this by site-directed mutagenesis of DNA that encodes for specific amino acids in NATs and KATs. The we test whether these changes alter their substrate specificity by utilizing the resulting novel proteins in a fluorescence-based enzymatic assay. The goal is to confirm the key elements that have been hypothesized to confer specificity of the enzymes and also determine how these elements differ between NATs and KATs.
We compare and contrast our new functioning versions of the NAT to an archaeal N-terminal acetyltransferase with the goal of altering the archaeal NAT to be more like our new NAT versions generated before, which contain properties of the original KAT and NAT. The archaeal NAT has a wide variety of substrates compared to other NATs. Using the same methods as above, we want to engineer a new version of the archaeal NAT that successfully acetylates both the N-terminal and lysine side chains, thereby producing the first acetyltransferase that is promiscuous enough to acetylate both substrates. By creating this “promiscuous” acetyltransferase, we will have created an acetyltransferase that could potentially become a molecular probe that could aid in the development of treatments of human diseases as well as allow us to infer what the original primordial acetyltransferase would have been like.
If you are interested in working at Dr. RC's lab please send an email to: yriveracolon@baypath.edu