Protein splicing: evidence for an N-O acyl rearrangement as the initial step in the splicing process.
Protein splicing involves the self-catalyzed formation of a branched intermediate, which then resolves into the excised intervening sequence and the spliced protein. A possible mechanism for branched intermediate formation is an N-O rearrangement of the peptide bond involving the amino group of the conserved serine/cysteine residue at the upstream splice junction to yield a linear peptide ester intermediate. This possibility was examined in using an in vitro splicing system involving the intervening sequence from the DNA polymerase of the extremely thermophilic archeon, Pyrococcus sp. GB-D. Because thioesters react much more rapidly with nitrogen nucleophiles at neutral pH than do oxygen esters, protein-splicing precursors in which the serine residue of interest was replaced by cysteine were constructed and purified. In the presence of 0.25 M hydroxylamine or 0.1 M ethylene diamine at pH 6 or higher, these constructs underwent rapid cleavage at the upstream splice junction, consistent with the aminolysis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage products. Comparison of the C-terminal peptide hydroxamate with the synthetic peptide hydroxamates with respect to chromatographic mobility, colorimetric assay, amino acid composition, and high-resolution mass spectrometry showed that the hydroxylamine-sensitive site in the splicing precursor was the peptide bond adjacent to the serine residue at the upstream splice junction. These results provide evidence that the peptide bond at the upstream splice junction can undergo a self-catalyzed N-O or N-S acyl rearrangement to yield a linear polypeptide ester intermediate and suggest that this kind of rearrangement constitutes the first step in protein splicing.
Shao
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Boston Biomedical Research Institute, Boston, Massachusetts 02114, USA.
Xu
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Paulus
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D000595:Amino Acid Sequence; D001105:Archaea / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D004952:Esters / Q000378:metabolism; D005029:Ethylenediamines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006877:Hydroxamic Acids / Q000032:analysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004952:Esters; D005029:Ethylenediamines; D006877:Hydroxamic Acids; D006898:Hydroxylamines; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019811:Hydroxylamine; C031234:ethylenediamine; D004259:DNA-Directed DNA Polymerase