Review articleStructure and function of xanthine oxidoreductase: where are we now?
Introduction
Xanthine oxidoreductase (XOR) is a complex molybdoflavoenzyme that is readily available from cows’ milk, where it forms a major component of the milk fat globule membrane (MFGM) [1], [2]. Largely because of this availability, XOR has been known for 100 years and studied in its essentially pure form for over 60 years [3]. The enzyme has consequently become a model for structural and mechanistic studies of molybdoenzymes in general, involving the application of electron spin resonance, x-ray absorption fine structure, and many other physicochemical techniques [4], [5]. The last few years have seen a dramatic increase in molecular information, with determination of its cDNA and gene sequences and publication of its three-dimensional structure.
XOR is generally recognized as the terminal enzyme of purine catabolism in man, catalyzing the hydroxylation of hypoxanthine to xanthine and of xanthine to urate. Until recently, little was known of the human enzyme, although inherited XOR deficiency, xanthinuria, has long been recognized, and, puzzlingly, known to be asymptomatic [6], [7]. Some 20 years ago, attention was directed toward the physiological and pathological significance of XOR by Granger, McCord, and colleagues, who proposed a role for XOR-derived reactive oxygen species (ROS) in ischemia-reperfusion (IR) injury [8], [9], [10]. Their hypothesis, which generated several hundred publications, undoubtedly helped to stimulate interest in XOR as a source of ROS, not only in many pathological states, but also in signal transduction generally. As recognition of the pathophysiological involvement of XOR increases, so does the need to understand its enzymology, kinetics, and control. As noted above, our structural knowledge of XOR has markedly increased in recent years. Moreover, a great deal of new information has become available concerning species and tissue variations of the enzyme, its substrate specificity, and regulation. For these reasons, it seems to be appropriate to review recent developments and to attempt to inter-relate them.
In general, the present survey focuses on works published in the last 10 years, and this is reflected in the reference list, which, wherever possible, covers earlier work by quoting more recent, especially review, articles. In view of the potential clinical relevance of much of the work described, emphasis is given to studies using mammalian, particularly human, enzyme and tissues.
Section snippets
Assays in tissues
XOR activity has been detected in all species examined, including bacteria [11], [12]. In mammalian tissues, activity is widely distributed, with highest levels being found in liver and intestine [12]. There is, however, considerable species variation, as exemplified by the wide range of levels in blood [12] and heart [13], [14]. In humans, apart from liver and intestine, most tissues show little XOR activity [12], [13], [14], [15], [16], [17]. XOR mRNA levels were also found to be highest in
Mammalian and avian XOR
The best-characterized form of XOR is undoubtedly that purified from bovine milk [1], [3]. XOR has also been purified from rat [42], [43], chicken [44], [45], and turkey [46] livers and from mouse mammary gland [47]. While these preparations are less well characterized than that from bovine milk, their properties are generally similar to those of the latter. In all cases, XOR occurs as a homodimer of approximately 300 kDa; each subunit contains four redox centers; viz. a molybdenum cofactor
Structure
The structure of the genetic loci coding for human [77] and mouse [78] XOR have been elucidated and the respective genes have been assigned to chromosomes 2p22 [79] and 17 [78]. These mammalian genes contain 36 exons and the exon-intron structure is highly conserved, differing greatly from those of the Drosophila or Calliphora genes, which are more compact, with only four or five exons [80].
Of mammalian and avian XORs, cDNA sequences have been reported for rat [81], mouse [82], chicken [83],
Ischemia-reperfusion injury
Granger and colleagues [8], [9], [10] focused attention on XOR by proposing a key role for the enzyme in the pathogenesis of ischemia-reperfusion (IR) injury. Their hypothetical mechanism, outlined in Fig. 4, can be briefly summarized as follows. In the course of ischemia, transmembrane ion gradients are dissipated, allowing elevated cytosolic concentrations of calcium. This, in turn, activates a protease that irreversibly converts XDH, predominant in vivo, into XO. Concurrently, cellular ATP
Circulating XOR
The presence of circulating XOR in mammals has long been recognized, and many different assays have been developed for its determination, particularly in humans, in which levels are particularly low. Most of these assays measure enzymic activity, and involve quantification of product by a range of means, including radioactivity (urate or xanthine) [127], [128], [129], fluorescence (isoxanthopterin) [130], [131], [132], chemiluminescence (superoxide) [133] and UV-visible spectroscopy (urate)
Pathological roles of XOR in the vasculature
Recent research into IR injury has focused on the vasculature, especially on leukocyte-endothelial cell interactions, which are relevant to many vascular diseases [122], [125], [126].
As noted in “Distribution,” XOR is present both in the cytoplasm and on the outer surface of endothelial cells [37], [38]. In the vasculature, such extracellular endothelial XOR could well be supplemented by circulating enzyme, levels of which can vary greatly in certain pathological states (see “Circulating XOR”).
Regulation
As noted in “Distribution,” XOR activity in human tissues is generally low, compared with other mammalian species. Clearly, enzyme activity is subject to control at several levels, more than one of which appear to be involved in this case. Thus, Xu and colleagues [195] have recently shown that, relative to mice, both transcription rates and core promoter activity of the human gene are repressed. Analysis of human XOR promoter activity in different cell types showed both repressor and activator
Enzymology
It has recently been established that XOR can act as a source of NO. Initial reports described the detection of NO, by chemiluminescence, following incubation of XOR with NADH and nitrate [227], [229] or nitrite [228], [229], [230] under hypoxic conditions. The anaerobic XOR-catalyzed reduction of inorganic nitrite to NO was subsequently investigated in detail by Godber et al. [182], who showed xanthine to be a more effective reducing substrate than NADH in this reaction. Steady state kinetic
Conclusion
As noted in the “Introduction,” interest in XOR received a major boost in the 1980s, following publication of its hypothetical involvement in IR injury. In fact, and not only for this reason, a great deal of new information concerning the enzyme has been gained in the last 20 years. Nevertheless, as will be clear from this review, many uncertainties remain.
Studies of the tissue and cellular distribution of XOR are contradictory and potential reasons for this are discussed in “Distribution.”
Acknowledgements
I am most grateful to Dr. Arwen Pearson for the simulated structures of Fig. 3 and to Jonanthan Gledhill for help with other figures. Thanks are also due to Prof. Robert Eisenthal for discussions and careful reading of the manuscript.
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Roger Harrison obtained an MA in Natural Sciences at the University of Cambridge and a PhD in Chemistry at the University of Birmingham, UK. Following postdoctoral research fellowships at the State University of New York, Buffalo, NY, USA, and at the National Institutes of Health, Bethesda, MD, USA, he was awarded an Alexander von Humboldt research fellowship at the University of Hamburg, Germany, where he spent 2 years. After a final postdoctoral research fellowship at the University of Loughborough, UK, he joined the University of Bath, where he is presently Professor of Biochemistry. For the last 12 years his research group has focused on the enzymology and immunology of xanthine oxidoreductase, with particular interest in the human enzyme, and more latterly on its catalysis of nitric oxide and peroxynitrite production.