Our aim was to investigate the mechanism of sulfur replacement by selenium in the 5-methylaminomethyl-2-thiouridine moiety of tRNA in vitro using partially purified enzyme preparations from Salmonella typhimurium. Several types of column chromatography were used to purify the enzyme(s) required for this process. The column fractions (even when combined) did not show the ability of the broken cell preparations to synthesize 5- methylaminomethyl-2-selenouridine from the sulfur analog present in tRNAs suggesting that an enzyme component was lost upon purification. Although the mechanism of the selenium substitution and the enzymes required have not been elucidated, it is known that mutation of a single gene, selD, prevents incorporation of selenium into tRNAs. In the present work we have studied the reaction catalyzed by the SELD enzyme (the product of selD gene) because revealing the products of the SELD enzyme reaction may help in purifying the other enzyme(s) needed for the biosynthesis of selenouridines in tRNAs. It would allow to complement the column fractions with the SELD enzyme product which probably serves as a selenium donor compound in the biosynthesis of 5-methylaminomethyl-2-selenouridine. Our 31P NMR studies showed that the products of this reaction are AMP, orthophosphate and selenophosphate. A mutant Salmonella strain (selA1) carrying a defective selD gene fails to catalyze selenium incorporation into tRNA. We used broken cell preparations from selA1 mutant cells complemented with purified SELD enzyme to find out whether the other enzyme(s) required for the selenium incorporation into tRNAs are active in this mutant cell preparation. We have found that supplementation of the deficient enzyme preparation with the purified SELD protein restored synthesis of seleno-tRNAs. We have also found that selenophosphate, the product of the SELD protein, can be substituted for the SELD enzyme in the selenium incorporation reaction.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Intramural Research (Z01)
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National Heart, Lung, and Blood Institute
United States
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