Selenium cofactor

Figure 20-3. Role of the pentose phosphate pathway in the glutathione peroxidase reaction of erythrocytes. (G-S-S-G, oxidized glutathione G-SH, reduced glutathione Se, selenium cofactor.)...

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). 

The vast majority of research focused on selenium in biology (primarily in the fields of molecular biology, cell biology, and biochemistry) over the past 20 years has centered on identification and characterization of specific selenoproteins, or proteins that contain selenium in the form of selenocysteine. In addition, studies to determine the unique machinery necessary for incorporation of a nonstandard amino acid (L-selenocysteine) during translation also have been central to our understanding of how cells can utilize this metalloid. This process has been studied in bacterial models (primarily Escherichia colt) and more recently in mammals in vitro cell culture and animal models). In this work, we will review the biosynthesis of selenoproteins in bacterial systems, and only briefly review what is currently known about parallel pathways in mammals, since a comprehensive review in this area has been recently published. Moreover, we summarize the global picture of the nonspecific and specific use of selenium from a broader perspective, one that includes lesser known pathways for selenium utilization into modified nucleosides in tRNA and a labile selenium cofactor. We also review recent research on newly identified mammalian selenoproteins and discuss their role in mammalian cell biology. 

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... 

NAH is composed of four subunits (SDS-PAGE) and contains a molybdenum cofactor (Dilworth 1983). Analysis of the electron paramagnetic resonance (EPR) spectra of the molybdenum center of NAH revealed a coordination of molybdenum to selenium (Gladyshev et al. 1994b). Apparently NAH is much like other selenium-dependent molybdenum hydroxylases such as XDH from C. barkeri and other purinolytic Clostridia. Whether or not the selenium is present as a ligand of molybdenum or is coordinated to molybdenum while being bound to another molecule (e.g., sulfur of cysteine) is still not known. The nature of the selenium cofactor and the mechanism of its incorporation into NAH are most likely similar to XDH and thus also require more study. 

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. 

Trace elements are essential cofactors for numerous biochemical processes. Trace elements that are added routinely to PN include zinc, selenium, copper, manganese, and chromium. There are various commercial parenteral trace element formulations that can be added to PN admixtures (e.g., MTE-5 ). Zinc is important for wound healing, and patients with high-output fistulas, diarrhea, burns, and large open wounds may require additional zinc supplementation. Patients may lose as much as 12 to 17 mg zinc per liter of gastrointestinal (GI) output (e.g., from diarrhea or enterocutaneous fistula losses) however, others have demonstrated that 12 mg/day may be adequate to maintain these patients in positive zinc balance.18 Patients with chronic diarrhea, malabsorption, and short-gut syndrome may have increased selenium losses and may require additional selenium supplementation. Patients with severe cholestasis should have copper and manganese... 

Probably the most effective use of XRF and TXRF continues to be in the analysis of samples of biological origin. For instance, TXRF has been used without a significant amount of sample preparation to determine the metal cofactors in enzyme complexes [86]. The protein content in a number of enzymes has been deduced through a TXRF of the sulfur content of the component methionine and cysteine [87]. It was found that for enzymes with low molecular weights and minor amounts of buffer components that a reliable determination of sulfur was possible. In other works, TXRF was used to determine trace elements in serum and homogenized brain samples [88], selenium and other trace elements in serum and urine [89], lead in whole human blood [90], and the Zn/Cu ratio in serum as a means to aid cancer diagnosis [91]. 

Figure 1 Overview of specific use of seienium in bioiogical systems. Selenium can be incorporated into macromolecules in at least three separate pathways. From the reduced form of selenide, selenium is activated to selenophosphate by the action of the enzyme selenophosphate synthetase (SPS or SelD). This activated form is then used as a substrate for pathway-specific enzymes that lead to (1) insertion as selenocysteine into proteins during translation (selenoproteins), (2) incorporation into tRNA molecules as mnm Se U or Se U, and (3) insertion into a unique class of molybdoenzymes as a labile, but required, cofactor. The need for activation to selenophosphate has been demonstrated in all cases at the genetic and biochemical level, with the exception of the labile selenoenzymes, where activation of selenium has only been proposed based on proximity of genes within an operon encoding SPS and a molybdoenzyme. ...

The first biochemical analysis of a selenium-containing XDH was reported in 1999 by Andreesen s group. This preparation was specific for xanthine and did not hydroxylate nicotinic acid. Moreover, the enzyme contained FAD, acid-labile sulfur, iron, and a dinucleotide molybdenum cofactor. Most intriguing was the near-equimolar presence of tungsten and molybdenum. It should be noted that the culture medium contained nearly equimolar levels of these metals, making one wonder whether the specificity of this enzyme for metal may be relaxed (i.e., can use Mo or W). Selenium was also found in the preparation and could be released by treatment with cyanide indicating it was also a labile cofactor. This further confirmed the chemical nature of the cofactor from the NAH enzyme from the same strain. ... 

Comparative genomics reveals potential widespread use of selenium in the form of a labile cofactor... 

Another study (Self and Stadtman 2000) described the purification of XDH from C. purinolyticum, a purine-fermenting strain originally isolated as an adenine-fermentor (Dtirre et al. 1981). Selenium was labile (cyanolyzable) and required for XDH activity. Similar to XDH from E. barkeri, XDH from C. purinolyticum consisted of three subunits (determined by SDS-PAGE). However, XDH from C. purinolyticum was significantly stable when isolated under aerobic conditions. Although these reports solidify the previously speculative data that selenium is present as a labile cofactor in clostridial XDH, the exact nature of selenium and the molecules to which... 

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. 

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... 

Nicotinic acid hydroxylase from Clostridium barkerii catalyzes reaction (55), the hydroxylation of a pyridine group, and has similarities to xanthine dehydrogenase. Nicotinic acid hydroxylase is a 300 000 molecular weight flavoprotein containing iron-sulfur and FAD centres, selenium1034 and a molybdopterin cofactor.1035 Formate dehydrogenase contains selenium as selenocysteine,1036 but this does not appear to be the case for nicotinic acid hydroxylase. The possibility that the selenium is incorporated into the molybdopterin cannot be excluded at present. 

Carbon monoxide serves as the sole carbon and energy source for the carboxydo bacteria under aerobic conditions. Using water as the oxygen donor, carbon monoxide oxidase catalyzes the hydroxylation of carbon monoxide, giving carbon dioxide or bicarbonate for assimilation. Most work has been carried out on the enzyme from Pseudomonas carboxydovorans.,ftJ7>W38 The activity of carbon monoxide oxidase is considerably stimulated upon anaerobic treatment with sulfide and dithionite, or by aerobic treatment with selenite. The binding of selenite to the oxidase specifically activates the CO — methylene blue reaction.1039 The molybdenum cofactor liberated from selenium-activated carbon monoxide oxidase does not contain selenium. Here, then, the... 

The results described in the previous sections have demonstrated the versatility of molybdenum as a reaction centre in biology. The molybdenum cofactor stands at the centre of an important network of cellular functions that are all catalyzed by molybdoenzymes. The similarities and differences in these reactions are of great interest, and relate clearly to the detailed arrangement of terminal sulfur and oxygen atoms. However, the unexpected results found for carbon monoxide oxidase and the requirement for selenium in some cases indicate that other factors are also important. 

An enzyme cofactor can be either an inorganic ion (usually a metal cation) or a small organic molecule called a coenzyme. In fact, the requirement of many enzymes for metal-ion cofactors is the main reason behind our dietary need for trace minerals. Iron, zinc, copper, manganese, molybdenum, cobalt, nickel, and selenium are all essential trace elements that function as enzyme cofactors. A large number of different organic molecules also serve as coenzymes. Often, although not always, the coenzyme is a vitamin. Thiamine (vitamin Bj), for example, is a coenzyme required in the metabolism of carbohydrates. 

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