Solution of the crystal structure of human milk xanthine oxidoreductase

Abstract

This thesis reports the crystallization, structure solution and preliminary refinement of the demolybdo form of human milk XOR to a resolution of 3.5 A. Although the bovine milk XOR structure was also recently determined, this is the first demolybdo XOR structure to be determined and also the first structure of a human XOR. Also reported is the determination of the cDNA sequence of human mammary gland XDH. Xanthine oxidoreductase is a complex molybdenum and iron-sulphur containing flavoprotein. Due to its abundance, the enzyme from bovine milk has been extensively studied over the last 100 years. More recently the human milk enzyme has been investigated and has been shown to have surprising properties, in particular a very low specific activity. Traditionally associated with the last stages of purine catabolism, XOR has recently been shown to produce reactive oxygen species through reduction of oxygen and also to be capable of reducing nitrates and nitrites to NO. These findings implicate XOR in a variety of disease states, in particular ischaemia reperfusion injury. The low specific activity of human milk XOR is due to a low content of molybdenum (~ 5 %). Higher molybdenum content and specific activity has been reported for certain other human tissues, notably liver and small intestine. This work aimed to investigate possible reasons for the low level of incorporation of molybdenum and its associated molybdopterin cofactor into the human milk enzyme.