Selenium-dependent enzymes from molybdenum hydroxylase

Gladyshev VN, SV Khangulov, TC Stadtman (1994) Nicotinic acid hydroxylase from Clostridium barkeri electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc Natl Acad Sci USA 91 232-236. 

Another selenium-containing molybdenum hydroxylase that has been isolated from Clostridium barkeri (identical to Eubacterium barkeri) is nicotinic acid hydroxylase (NAH). Clostridium barkeri was isolated initially as a fermentor of nicotinic acid and thus NAH is a key enzyme in the efficient fermentation of nicotinic acid as a source of carbon and energy. NAH contained selenium when purified from cells labeled with Se-selenite. However, this label was lost during denaturing gel electrophoresis and also on heating of the enzyme (Dilworth 1982). Exhaustive analysis of selenium-labeled alkylation products of NAH under various conditions revealed selenium was bound as a labile cofactor (Dilworth 1982), and not as seleno-cysteine. This report was the first to describe a selenium-dependent enzyme that did not contain selenium in the form of selenocysteine. 

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. 

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