New approaches for interpreting protein molecular docking data of structurally complex substrates and promiscuous enzymes
By: Cillian Variot and Daniel Capule
Department: Biochemistry
Faculty Advisor: Dr. Misty L. Kuhn
Gcn-5 related N-acetyltransferase (GNAT) enzymes are prevalent in diverse organisms and catalyze the transfer of an acyl group from a donor molecule to the primary amine of an acceptor substrate. The superfamily is characterized by a structurally conserved splay of a beta-sheet motif between B4 and B5 strands and varying acceptor pockets that allow them to acetylate a breadth of substrates. Less is known about acceptor pockets of GNATs, especially which residues are important for acceptor substrate recognition. Therefore, we selected a promiscuous enzyme PA3944 from Pseudomonas aeruginosa to better understand the importance of acceptor site residues for substrate recognition and specificity/promiscuity. Our previous studies with PA3944 have shown that it can acetylate a variety of substrates including polymyxin antibiotics through a hybrid ping-pong mechanism. We also found the enzyme facilitates acetyl transfer through a nucleophilic serine (S148) within the acceptor pocket. To further explore substrate binding in the PA3944 acceptor site, we performed molecular docking studies. Additionally, we used acetylated and nonacetylated S148 models of the enzyme in the presence and absence of CoA to simulate in-vitro forms during the acetyl transfer reaction. Given the sheer volume and complexity of computational results generated by molecular docking, we developed a method to sort docking results by proximity to the carbonyl carbon of the acetyl donor. We paired these results with our in vitro kinetic studies of mutated acceptor pocket residues and found the residue interactions of docked molecules are substrate specific.