Date of Award
Mycobacterium ulcerans is the causative agent of Buruli ulcer, a tropical skin disease that affects thousands of individuals annually. Recent studies have revealed that lipolytic enzymes are involved in the pathogenicity processes of mycobacterium and could be potential targets for novel antibiotics. LipN is one proposed serine hydrolase in Mycobacterium ulcerans that contains the conserved α/β hydrolase protein fold and utilizes the conserved catalytic traid of serine, histidine, and aspartate/glutamate. The physiological substrate and biological role of LipN from M. ulcerans have not yet been determined. In this study, LipN was cloned into a pET28a plasmid and overexpressed in an E. coli host. Ni-affinity chromatography was used to purity LipN from the E. coli host cell lysate. The substrate specificity of LipN was elucidated using enzymatic assays utilizing a library of 15 latent fluorophore substrates. Steady state kinetic data was fit to the Michaelis-Menten equation; kcat, KM, kcat/KM were derived to quantitate the structure-activity relationships that provide insight into the substrate specificity of LipN. The LipN protein showed preference for 2-4 carbon chains with the highest catalytic efficiency (kcat/KM of 881±64 M-1s-1) for an alky ether ester substrate.
Raynor, Stephanie, "Substrate Specificity of the LipN Hydrolase from Mycobacterium ulcerans" (2016). Undergraduate Honors Thesis Collection. 358.