The structure of protein molecules : in celebration of the International Year of Crystallography, 2014

Many people, including laymen, are aware of the double helical nature of the DNA molecule. A few may actually realise that it was the technique of X-ray crystallography that was the key to solving this structure. Even fewer will understand the uses and applications of crystallography to the most diverse of biological materials; proteins. In this review we discuss the application of a number of methodologies required to progress from a cloned gene to protein expression and purification, crystallisation conditions and eventually to X-ray structure determination. We provide our own experience in the field as examples of the procedures required. Protein crystallographers worldwide are contributing to our understanding of how enzymes work, how our immune system defends us against viruses and are using structural information to design novel pharmaceutical reagents.peer-reviewe

Role of protein structure in drug discovery

Many pharmaceuticals currently available were discovered either during the screening of natural of synthetic product libraries or by serendipitous observation. Such a \random" approach entails testing numerous compounds and developing countless high-throughput screening assays. On the other hand, a "rational" approach involves the structure-based route to drug discovery, where the structure of a target protein is determined. Hypothetical ligands may be predicted by molecular modelling, while movement of a molecule may be predicted by Molecular Dynamics Simulations prior to synthetic chemical synthesis of a particular molecule. Here, we will be discussing protein structure-based approaches to drug discovery.peer-reviewe

The Good and the Bad: The Bifunctional Enzyme Xanthine Oxidoreductase in the Production of Reactive Oxygen Species

Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme which occurs in two forms; the reduced form known as xanthine dehydrogenase (XDH, EC 1.17.1.4) and the oxidised form known as xanthine oxidase (XO, EC 1.17.3.2). In humans, it is a 293 kDa homodimer which catalyses consecutive hydroxylation steps of purine degradation. The oxidised form of the enzyme produces hydrogen peroxide and superoxide (O2•−), both of which are reactive oxygen species (ROS) that can interact with several biomolecules producing adverse reactions. XOR can also produce nitric oxide, a cardiovascular protective molecule. Overproduction of nitric oxide results in the formation of the highly reactive peroxynitrite radical. XOR-produced ROS may provide protection against infection, while at the same time can also lead to inflammation, oncogenesis, brain injury and stroke. XOR is also involved in tumour lysis syndrome in chemotherapy patients as well in ischaemia-reperfusion injury, increasing the levels of ROS in the body. Consequently, the presence of XOR in blood can be used as a biomarker for a number of conditions including oxidative stress and cardiovascular disease

Purification and characterization of xanthine oxidoreductases from local bovids in Malta

Xanthine oxidoreductase (XOR) is a molybdoflavoprotein mainly involved in purine catabolism. It exists in two forms, the oxidase (XO) and dehydrogenase (XDH) which are inter-convertible within mammalian cells. Although various researchers have reported the extraction of mammalian XOR, no extractions have yet been carried out in Malta and subsequently no characterizations are available. In this study, XOR was successfully purified from bovine, caprine and ovine milk through a multistep purification process involving both chemical and chromatographic techniques. The molecular weights of the native enzyme were found to be 295 kDa, 281 kDa and 275 kDa, representing the bovine, caprine and ovine XOR respectively. Western blot showed XOR to be represented on SDS-PAGE by a minimum of three major bands having molecular weights of 151 kDa, 131 kDa and 85 kDa. While all samples showed activity on native PAGE, spectrophotometric assays revealed the bovine XOR to be the most active. Surprisingly, the addition of NAD+ to the assay mixture inhibited enzyme activity of the bovine and caprine XOR whereas the ovine XOR doubled its activity in response to NAD+. The latter also showed a lower binding affinity to heparin. Following incubation with trypsin, XOR was irreversibly converted to its oxidase form in all samples as reflected by the observed increase in XO activity.peer-reviewe

IntecOpen

Xanthine oxidoreductase (XOR) is a molybdoflavin enzyme which occurs in two forms; the reduced form known as xanthine dehydrogenase (XDH, EC 1.17.1.4) and the oxidised form known as xanthine oxidase (XO, EC 1.17.3.2). In humans, it is a 293 kDa homodimer which catalyses consecutive hydroxylation steps of purine degradation. The oxidised form of the enzyme produces hydrogen peroxide and superoxide (O2 •−), both of which are reactive oxygen species (ROS) that can interact with several biomolecules producing adverse reactions. XOR can also produce nitric oxide, a cardiovascular protective molecule. Overproduction of nitric oxide results in the formation of the highly reactive peroxynitrite radical. XOR-produced ROS may provide protection against infection, while at the same time can also lead to inflammation, oncogenesis, brain injury and stroke. XOR is also involved in tumour lysis syndrome in chemotherapy patients as well in ischaemia-reperfusion injury, increasing the levels of ROS in the body. Consequently, the presence of XOR in blood can be used as a biomarker for a number of conditions including oxidative stress and cardiovascular disease.peer-reviewe