Escherichia coli mutant SELD enzymes. The cysteine 17 residue is essential for selenophosphate formation from ATP and selenide

Synthesis of a labile selenium donor compound, selenophosphate, from selenide and ATP by the Escherichia coli SELD enzyme was reported previously from this laboratory. From the gene sequence, SELD is a 37-kDa protein that contains 7 cysteine residues, 2 of which are located at positions 17 and 19 in the sequence -Gly-Ala-Cys-Gly-Cys-Lys-Ile- (Leinfelder, W., Forchhammer, K., Veprek, B., Zehelein, E., and Bock, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 73, 543-547). Inactivation of the enzyme by alkylation with iodoacetamide indicated that at least 1 cysteine residue in the protein is essential for enzyme activity. To test the possibility that the Cys¹⁷ and/or Cys¹⁹ residue might be essential, these were changed to serine residues by site-specific mutagenesis. The biological activities of the wild type and mutant proteins were studied using E. coli MB08 (selD^-) transformed with plasmids containing the selD genes. The plasmid containing the Cys¹⁷- mutated gene failed to complement MB08, whereas the Cys¹⁹-mutated gene was indistinguishable from wild type. The mutant proteins, like the wild type enzyme, bound to an ATP-agarose matrix, showing that their affinities for ATP were unimpaired. Selenide-dependent formation of AMP from ATP was abolished by mutation of Cys¹⁷, but the Cys¹⁹ mutation had no effect on the ability of the enzyme to catalyze the reaction. These results indicate that Cys¹⁷ has an essential role in the catalytic process that leads to the formation of selenophosphate from ATP and selenide.