Abstract 1554 Purification and Crystallization of Thermostable Acetyl Esterase Mutants

Serine hydrolases are a large category of enzymes that play vital roles in all forms of life. In humans, they can serve as targets for drugs and the building blocks in the development of new antibiotics. Serine hydrolases can be further subclassified into a group called carbohydrate esterase family 7 (CE7) which hydrolyze ester bonds of carbohydrate substrates. This project focuses on TM0077, an acetyl esterase isolated from Thermotoga maritima, which is a specific enzyme that is a member of the CE7 family and uses a catalytic triad to hydrolyze acetyl ester functional groups of carbohydrate substrates. Previous studies have determined conserved proline and isoleucine residues in the active site of TM0077 that play a large role in the conformation of the active site and thus, the substrates that the active site can accompany. Different mutations of these two residues in TM0077 are constructed and the protein is expressed in LOBSTR E. Coli cells and purified. These proteins are crystallized, and their structure will be determined using X-ray crystallography. The effect that these mutations have on TM0077's three-dimensional structure and substrate binding will be analyzed.