Structural and functional insights of human 5-lipoxygenase

摘要: This work describes the structure of human 5-lipoxygenase (5-LOX)(Gilbert, Bartlett et al. 2011) and the techniques required to ascertain the structure. 5-LOX is a notoriously unstable enzyme with important biological functions. The human 5-LOX has been implicated in many disease states including asthma, atherosclerosis and cancer. Part of 5-LOX biology is the inherent instability of the enzyme, which is thought to help regulate the production of its pro-inflammatory products, the leukotrienes. Our objective was stabilizing the enzyme for in vitro studies without affecting catalytic fidelity. I was able to quantitate stabilizing and destabilizing point mutations of 5-LOX by thermal denaturation and kinetic analysis along with other biochemical techniques. Through rigorous site-directed mutagenesis experiments and multiple expression protocols, I was able to over-express an engineered form of 5-LOX that is soluble and …