An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site [Protein Structure and Folding]
October 9th, 2015 by Bachmann, A.-L., Mootz, H. D.
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 up-hill 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 IntN fragment could be shortened from 37 aa to 28 aa and exchanged with the 25 aa IntN fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the IntC fragment of the GOS-TerL intein.