title: An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site.
abstract: Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine, or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal uphill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N terminus. The Int(N) fragment could be shortened from 37 to 28 amino acids and exchanged with the 25-amino acid Int(N) fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the Int(C) fragment of the GOS-TerL intein.
authors.affiliation: From the Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Muenster, 48149 Münster, Germany.
authors.forename: Anne-Lena
authors.initials: AL
authors.lastname: Bachmann
authors.affiliation: From the Department of Chemistry and Pharmacy, Institute of Biochemistry, University of Muenster, 48149 Münster, Germany Henning.Mootz@uni-muenster.de.
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
mesh_terms: D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D030561:Databases, Nucleic Acid; D047668:Inteins / Q000502:physiology*; D054892:Metagenome; D017354:Point Mutation; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D012694:Serine; D003545:Cysteine
keywords: biotechnology; intein; metagenomics; post-translational modification (PTM); protein splicing; site-directed mutagenesis; structural model