Purine hydroxylase

Self WT (2002) Regulation of purine hydroxylase and xanthine dehydrogenase from Clostridium purinolyti-cum in response to purines, selenium and molybdenum. J Bacterial 184 2039-2044. 

Although it had been assumed that only hypoxanthine dehydrogenase is required for the conversion of hypoxanthine (6-hydroxypurine) into uric acid, in Clostridium purinolyti-cum, two enzymes, both of which contain a selenium cofactor, are required. The enzymes differ in the molecular mass of their subunits, in their terminal amino acid sequences, in their kinetic parameters, and in their specific activities for purines (Self and Stadman 2000). Purine hydroxylase converts purine into hypoxanthine and xanthine (2,6-dihy-droxypurine), which is then further hydroxylated to uric acid (2,6,8-trihydroxypurine) by xanthine dehydrogenase (Self 2002). 

Self WT, TC Stadman (2000) Selenium-dependent metabolism of purines a selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized. Proc Natl Acad Sci USA 97 7208-7213. 

Upon purification of the XDH from C. purinolyticum, a separate Se-labeled peak appeared eluting from a DEAE sepharose column. This second peak also appeared to contain a flavin based on UV-visible spectrum. This peak did not use xanthine as a substrate for the reduction of artificial electron acceptors (2,6 dichlor-oindophenol, DCIP), and based on this altered specificity this fraction was further studied. Subsequent purification and analysis showed the enzyme complex consisted of four subunits, and contained molybdenum, iron selenium, and FAD. The most unique property of this enzyme lies in its substrate specificity. Purine, hypoxanthine (6-OH purine), and 2-OH purine were all found to serve as reductants in the presence of DCIP, yet xanthine was not a substrate at any concentration tested. The enzyme was named purine hydroxylase to differentiate it from similar enzymes that use xanthine as a substrate. To date, this is the only enzyme in the molybdenum hydroxylase family (including aldehyde oxidoreductases) that does not hydroxylate the 8-position of the purine ring. This unique substrate specificity, coupled with the studies of Andreesen on purine fermentation pathways, suggests that xanthine is the key intermediate that is broken down in a selenium-dependent purine fermentation pathway. ... 

In addition to the molybdenum hydroxylases mentioned above, a new selenium-dependent hydroxylase with specificity for purine and hypoxan-thine as substrates, termed purine hydroxylase, was uncovered during purification of XDH from C. purinolyticum (Self and Stadtman 2000). Purified PH was labeled with Se and was not reduced in the presence of xanthine as a substrate. As with other selenium-dependent molybdenum hydroxylases, selenium was removed by treatment with cyanide with parallel loss in catalytic activity. Selenium was also efficiently removed in the presence of low ionic strength buffer during final dialysis of PH, indicating that ionic strength affects the stability of the labile selenium cofactor in this enzyme. 

Fe2S2] clusters are part of the molybdenum containing hydroxylases. Typically, apart from molybdenum and two EPR-distinct iron-sulfur centres there can be FAD as additional cofactor. In Chlostridium purinolyticum a selenium-dependent purine hydroxylase has been characterized as molybdenum hydroxylase. The EPR of the respective desulfo molybdenum (V) signal indicated that the Mo-ligands should differ from those of the well known mammalian corollary xanthine oxidase.197 For the bacterial molybdenum hydroxylase quinoline oxidoreductase from Pseudomonas putida an expression system was developed in order to be able to construct protein mutants for detailed analysis. EPR was used to control the correct insertion of the cofactors, specifically of the two [Fe2S2] clusters.198... 

A purine hydroxylase from fungi,639 bacterial quinoline and isoquinoline oxidoreductases,640/641 and a selenium-containing nicotinic acid hydroxylase from Clostridium barberei6i2 are members of the... 

As indicated in Fig. 25-18, free adenine released from catabolism of nucleic acids can be deaminated hydrolytically to hypoxanthine, and guanine can be deaminated to xanthine.328 The molybdenum-containing xanthine oxidase (Chapter 16) oxidizes hypoxanthine to xanthine and the latter on to uric acid. Some Clostridia convert purine or hypoxanthine to xanthine by the action of a selenium-containing purine hydroxylase.3283 Another reaction of xanthine occurring in some plants is conversion to the trimethylated derivative caffeine. 328b One of the physiological effects of caffeine in animals is inhibition of pyrimidine synthesis.329 However, the effect most sought by coffee drinkers may be an increase in blood pressure caused by occupancy of adenosine receptors by caffeine.330... 

Figure 3.5 Plots of the -values of Mo(v) species against g -anisotropy for members of the XO enzymes family. In red, slow-type signals from Dg AOR slow-type aquo from milk XO (2), with %poxanthine from Clostridium purinolyticum Purine Hydroxylase (3). In black, rapid-type signal from milk XO (4), rapid q from Pseudomonas putida Quinoline 2-Oxidore-ductase (5), rapid type 1 with 1-methybcanthine from milk XO (6), Thauera aromatica 4-Hydroxybenzoyl-CoA reductase (7), rapid q from As QualOx (8), rapid type 2 borate from milk XO (9), resting q2 from Pp QuinOr (10), rapid q from Pd IsoOr (11), resting q from Pp QuinOr (12). In blue, very rapid-type signals from Pp QuinOr (13), with 2-Hydroxy-6-methylpurine from milk XO (14)," from Pd IsoOr (15), from As QualOx (16). Linear correlations between the available data sets within each family can be extrapolated and are schematically indicated by straight lines to help the reader.

Different members of the xanthine oxidase family show differing susceptibility to cyanide. Thus, while mammalian aldehyde oxidases react more readily with cyanide, purine hydroxylase II imm Aspergillus nidulans was found to be highly resistant and only with extensive treatment did the desulfo form develop. Early XAS work indicated the presence of an oxo-sulfido active site with both Mo=0 and Mo=S coordination, and that the cyanolyzable sulfur is the Mo=S, and resolving a debate in which Mo=S and persulfide were candidates for the cyanolyzable sulfur. Xanthine oxidase also reacts with arsenite As(OH)3 to form an inhibited complex in which molybdenum is bound by arsenic through the labile sulfur (Mo-S-As). The conclusions of the early XAS study have recently been confirmed and extended by protein crystallography of the inhibitory species. ... 

Cytochrome oxidase Pro)>ioiiic acid oxidase Choline oxidase Iliotiii oxidase Sulfur oxidases Nitroethanc oxidase Cholesterol dehydrogenase Purine hydroxylase Fe Porphyrin Cytochrome c 4-(Cu ) H,0 UNCLASSIFIED OXIDASES 293 824 607 32 257 494 689 44... 

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