Identification of an unusual intein in chloroplast ClpP protease of Chlamydomonas eugametos.
The proteasome-like ClpP protease is widely distributed and structurally conserved among bacteria and eukaryotic cell organelles. In Chlamydomonas eugametos, however, the chloroplast clpP gene predicted a much larger ClpP protein containing large insertion sequences (ISs). One insertion sequence, IS2, is 456 amino acid residues long and not similar to known proteins. Here we show that IS2 is an unusual intein, and its protein splicing activity in Escherichia coli cells can be activated by a single amino acid substitution. Analysis of IS2 sequence revealed short sequence motifs that are similar to known intein motifs, including putative LAGLI-DADG endonuclease motifs. But a histidine residue conserved at the C terminus of known inteins is replaced in the IS2 sequence by a glycine residue (Gly455), rendering the IS2 sequence incapable of detectable protein splicing when tested in E. coli cells. Changing Gly455 to histidine activated the ability of IS2 to undergo protein splicing in E. coli cells. The IS2 sequence (intein) was precisely excised from a precursor protein, with the flanking sequences (exteins) joined together by a normal peptide bond.
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Biochemistry Department, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
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D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000235:genetics; D002696:Chlamydomonas / Q000201:enzymology* / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases