Structure and Activity of Inteins from Pyrococcus abyssi and Pyrococcus horikoshii

Abstract

Protein splicing is the self‐catalyzed excision of an intervening polypeptide (intein) from flanking polypeptides (exteins), concomitant with the ligation of the exteins. Similar inteins found in two thermophilic archaebacteria, Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho), interrupt the DNA Polymerase II. Comparison of the Pab PolII NMR structure and a Pho PolII homology model, along with 75% sequence identity between the inteins, reveals a common fold with the exception of a longer, disorganized loop in the Pab intein. The longer loop may provide a less rigid structure that allows splicing to occur at lower temperatures. Both splicing and uncoupled cleavage at the intein junctions occur to a greater extent at lower temperatures for the Pab intein than with the Pho intein. The relative rigidity of the inteins was compared by treatment of each with the thermostable protease thermolysin, which cleaves proteins in flexible regions. Thermolysin cleavage was more efficient at lower temperatures for the Pab intein. We are currently examining the relative strength of a disulfide bond that links the catalytic Cys residues as a measure of the flexibility of both inteins' active sites by titration with DTT.