The molybdoenzyme, xanthine oxidoreductase is a homodimer of 150 kDa subunits. It has wide substrate specificity but is best known for its capacity to oxidise ...
530s Biochemical Society Transactions (1997) 25 168 Xanthine oxidoreductase: dehydrogenase to oxidase conversion.
” BALENDRAN, MATTHEW G. RYAN, A ROGER HARRISON, ADRIAN WOLSTENHOLME, and GREGORY B. BULKL.EY*.
regarding the role of XDH pathophysiological processes.
The molybdoenzyme, xanthine oxidoreductase is a homodimer of 150 kDa subunits. It has wide substrate specificity but is best known for its capacity to oxidise hypoxanthine to xanthine and xanthine to uric acid; key stages in purine catabolism [l]. It exists in two forms, xanthine dehydrogenase (EC 1.1.1.204, XDH) and xanthine oxidase (EC 1.1.3.22, XO), which can be reversibly interconverted by sulphydryl reagents or irreversibly (XDH to XO) by proteolysis. XDH preferentially reduces NAD, in contrast to XO, which does not reduce NAD, passing its electrons instead to molecular oxygen. Reduction of oxygen leads to superoxide anion and hydrogen peroxide and it is the capacity to generate these reactive oxygen species (ROS) that has led to a great deal of interest in the enzyme as a pathogenic factor in ischaemia-reperhion injury [2]. ROS are also increasingly cited as intermediates in normal signal transduction [3]. The proposed mechanism of ischaemia-repefision injury [4] is as follows. During ischaemia, proteases are activated that cleave XDH ( predominant in vivo) to XO. Ischaemia also leads to breakdown of ATP to ADP and thence to hypoxanthine, which builds up in the tissues. On reperhion, oxygen becomes available and accepts electrons from the hypoxanthine- XO system, generating superoxide anion, hydrogen peroxide and subsequently more destructive forms of ROS. The XDH form of purified rat liver enzyme, can be proteolytically converted to the XO form by digestion with trypsin [5]. Three peptide fragments were produced, with molecular masses of 20, 40 and 85 kDa, corresponding to residues 1-184, 185-539 and 540-1319 respectively of the whole enzyme. Lysine residues 184 and 539 are clearly important in such proteolytic cleavage and we have sought to mutate these residues in order to assess the importance of XDH to XO conversion as a source of ROS in specific metabolic processes. Full length rat liver XOR cDNA ( generously supplied by Dr. Takeshi Nishino ) was subcloned into the mammalian expression vector pc DNA3 ( Invitrogen ). The putative sites of in vivo proteolysis, lysines 184 and 539, of the rat liver enzyme were mutated to the non-basic amino acids alanine and threonine respectively ( K184A and K539T ) using the Altered Sites II in vitro mutagenesis system ( Promega ). Plasmid pRX I PC designed for overexpression of the wild-type rat liver XOR gene was used to construct derivatives that expressed the mutant XOR, automated sequencing confirming the presence of the desired mutations. pAlter based phagemids carrying the rat sequence mutations K184A and K539T were digested with Not I / Xba I and Xba I / BstE I1 and the XOR fragments produced cloned into similarly digested pRX / PC to form three fill-length rat mutant expression plasmids pK184A, pK539T and pK184AK539T respectively. We have, accordingly, incorporated, into a mammalian expression vector, rat liver XOR cDNA, and also the mutated cDNA’s in which either one or both of the two lysines essential for tryptic cleavage have been replaced by non-basic amino acids. It is anticipated that expression of these four cDNA’s in appropriate mammalian tissue will provide valuable information
XO
conversion in
This work was supported by a programme grant from the National Institutes of Health. 1.
School of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK. *Johns Hopkins University, School of Medicine, 600 N. Wolfe St., Baltimore, Md 21287-3654, USA.
to
2. 3. 4. 5.
Bray, R. C.(1975) In: The Enzymes Vol XI1 3rd Ed. (ed. P. D. Boyer) pp 299-419. Academic Press, New York. Sussman, M. S.and Bulkley, G. B. (1990) Methods in Enzymol. 186, 71 1-723. Pahl, H. L. and Baeuerle, P. A. (1994) Bioessays 16, 497501. McCord, J. M. (1985) New Engl. J. Med. 312, 159-163. Amaya, Y., Yamazaki, K-i., Sato, M., Noda, K., Nishino, T., and Nishino, T. (1990) J. Biol. Chem. 265,14170-14175.
