Selenium-dependent enzymes from

Table 12.1. Selenium-dependent enzymes from Clostridiaceae.

Gladyshev VN, SV Khangulov, TC Stadtmann (1994) Nicotinic acid hydrolase from Clostridium barkeri electron paramagnetic studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. Proc Natl Acad 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. 

GSH and cysteine both have sulfhydryl groups that readily bind many phase I metabolites (Brown, 2001). GSH is a free-radical scavenger that prevents membrane damage from reactive metabolites. These reactions may be spontaneous or catalyzed by GSH peroxidases, which are selenium-dependent enzymes. Because these enzymes are cytosolic, damaged membrane phospholipids must... 

Imhoff D, JR Andreesen (1979) Nicotinic acid hydroxylase from Clostridium barkeri selenium-dependent formation of active enzyme. FEMS Microbiol Lett 5 155-158. 

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. ... 

One underlying question remains why does this small class of microorganisms require a labile selenium cofactor in these enzymes. Few have speculated on this in the published literature. Yet one key comparison between selenium and non-selenium-dependent hydroxylases may be quite telling. The well-studied bovine XDH has a turnover rate of approximately 5 while the PH enzyme from C. purinolyticum has a far... 

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. 

The hydrolysis, alcoholysis, and aminolysis of monoselenophosphate (294) have been reported for the first time (294) is the labile selenium donor compound required for the synthesis of Se-dependent enzymes and seleno-tRNAs, and is formed from ATP and selenide, HSe. The rate of hydrolysis of monoselenophosphate (294) is... 

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