Cloning and Expression of Bovine Xanthine Oxidase and Human
Xanthine oxidase (XO) is an enzyme that converts hypoxanthine to xanthine and xanthine to uric acid, the last two steps in purine metabolism of mammals. The mechanism is divided into a reductive and oxidative half reaction. XO uses oxygen as it final electron acceptor, thus, during ischemic conditions the oxidative half reaction does not occur resulting in electron build up. Upon reintroduction of oxygen, these reducing equivalents rapidly convert oxygen to H2O2 and O2? leading to oxidative tissue damage. Previous studies with a model enzyme, R. capsulatus xanthine dehydrogenase (RcXDH), have allowed comparative reactions with various mutations in the active site. Thus far RcXDH is the only enzyme of this type which has been successfully cloned and expressed in an E. coli system. In our project, we are attempting to clone and express both human XDH and bovine XO in and E. coli system. Expressed enzymes will be used to compare to previous data collected on RcXDH, confirm current mechanistic understanding, and determine binding orientation of various substrates and inhibitors. This information will then be used to develop an inhibitor which will decrease the rate of production and therefore subsequent damage from reactive oxygen species.