Amino acid sequence analysis of Escherichia coli formate dehydrogenase (FDHH) confirms that TGA in the gene encodes selenocysteine in the gene product

Abstract

The formate dehydrogenase (FDHF) of Escherichia coli is a selenocysteine-containing protein that occurs as a component of the formate-hydrogen lyase complex. The gene encoding this 80 kd polypeptide contains a TGA codon in the open reading frame. Several indirect lines of evidence showed earlier that the selenocysteine residue in the protein is inserted co-translationally in a TGA (UGA) dependent process. Direct proof that the selenocysteine is present in the polypeptide in the position corresponding to TGA as predicted from the gene sequence was obtained by automated amino acid sequence analysis of a 75Se-containing peptide isolated from the protein. Construction of a fusion gene comprising a small segment of the fdhF gene linked to the lacZ gene as reporter greatly facilitated isolation of the selenocysteine-containing protein. Subsequent cleavage of this isolated gene product with endoproteinase Asp-N gave rise to an easily purified small selenocysteine-containing peptide that was amenable to amino acid sequence analysis.