Title page for ETD etd-04202011-103203

Type of Document Dissertation
Author Gilbert, Nathaniel Carson
Author's Email Address ngilbe1@lsu.edu
URN etd-04202011-103203
Title Structural and Functional Insights of Human 5-Lipoxygenase
Degree Doctor of Philosophy (Ph.D.)
Department Biochemistry (Biological Sciences)
Advisory Committee
Advisor Name Title
Newcomer, Marcia Committee Chair
Bartlett, S Committee Member
Doerrler, W Committee Member
Lee, YH Committee Member
Day, Donald Dean's Representative
  • structure
  • arachidonic acid
  • 5-lipoxygenase
Date of Defense 2011-04-14
Availability unrestricted
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 stable for biochemical studies and most importantly amenable to crystallization. Our lab is the first to crystallize a human lipoxygenase and model the molecular details.

Another objective was studying the effect of phosphorylation on specific serine residues in 5-LOX that have been previously shown to be phosphorylated in vivo. I focused on phosphorylation of Ser663, which is ten amino acids removed from the C-terminus that penetrates the 5-LOX body and binds to the catalytic iron. I mutated this Ser663 to an aspartate, which is a mutation that has been previously shown to mimic a phosphorylated serine. The mutant 5-LOX S663D has altered product specificity in that it oxygenates arachidonic acid (AA) on the 15 carbon preferentially instead of the 5 carbon. This dual specificity of 5-LOX could alter the amounts of the pro- and/or anti-inflammatory compounds present at sites of activation.

With the structure of human 5-LOX solved and with reproducible crystal conditions, I began co-crystallization trials of 5-LOX with AA but devoid of oxygen, the other substrate. This would allow visualization of the important substrate binding amino acids and the catalytic machinery necessary for leukotriene production. I was able to model the placement of AA into the active site of Stable-5-LOX S663D, and this structure revealed striking conformational changes required for substrate binding. With these structural studies along with future co-crystallization studies with inhibitors, I will be able to describe amino acids important for catalysis. These insights afforded from co-crystal structures could aid in the development of therapies for the treatment of certain diseases caused by 5-LOX.

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