Purines molybdenum hydroxylases

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... 

Molybdenum Hydroxylases (Aldehyde Oxidase, Xanthine Oxidase) Oxidations Purines, pteridine, methrotrexate, quinolones, 6-deoxycyclovir... 

Other enzymes may also be involved in the oxidation of aldehydes, particularly aldehyde oxidase and xanthine oxidase, which belong to the molybdenum hydroxylases. These enzymes are primarily cytosolic, although microsomal aldehyde oxidase activity has been detected. They are flavoproteins, containing FAD and also molybdenum, and the oxygen incorporated is derived from water rather than molecular oxygen. Aldehyde oxidase and xanthine oxidase in fact oxidize a wide variety of substrates, both aldehydes and nitrogen containing heterocycles such as caffeine and purines (see below). Aldehyde... 

Both molybdenum hydroxylases can oxidize uncharged and cationic substrates, although at physiological pH aldehyde oxidase is much more active towards the latter group of compounds. It does not necessarily follow, however, that when compounds have a pKa value around 7, the neutral and protonated forms both bind to the enzyme. Kinetic studies at different pH values have indicated that it is the unprotonated form of 2-aminophthalazine that reacts with the molybdenum hydroxylases, either as a substrate for aldehyde oxidase or a competitive inhibitor of xanthine oxidase [211]. In contrast, at pH 7-8 many of the purine and pteridine substrates are present as mixtures of uncharged molecules and monoanions, both of which are rapidly oxidized by xanthine oxidase [181, 200, 206, 210, 212]. 

The biosynthesis of uric acid from the immediate purine precursor xanthine that results from adenine, via the intermediate hypoxanthine, or from guanine is illustrated in Figure 36.32. The enzyme xanthine oxidase (a molybdenum hydroxylase enzyme) is involved in two steps, the conversion of hypoxanthine to xanthine, and the final step, the conversion of xanthine to uric acid. Allopurinol originally was designed... 

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. 

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... 

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. ... 

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