Temperature and Pressure Dependence of the Activity of Inteins from Extreme Thermophiles

Abstract

Protein splicing is a post‐translational modification by which intervening polypeptides, or inteins, catalyze their own excision from two flanking polypeptides, or exteins, concomitant with the ligation of the exteins. We studied the activity of inteins that interrupt the DNA Polymerase II (PolII) from the deep‐sea thermophiles Thermococcus kodakarensis (Tko) and Thermococcus barophilus (Tba). Previous work with Pyrococcus abyssi (Pab) and Pyrococcus horikoshii (Pho) PolII inteins, which both possess flexible loop regions, showed temperature‐dependent splicing activity. The Tba and Tko inteins we studied, on the other hand, contain a homing endonuclease region in place of this flexible loop. We found that, unlike the Pab and Pho PolII inteins, the Tba and Tko PolII inteins splice poorly in E. coli, but can be induced to splice in vitro at higher temperatures. Mutagenesis studies suggest that changing the Tba PolII intein's native final reside (Asn to Gln) slowed splicing significantly, as did changing the native penultimate Ser to His. Mutating the Tko PolII intein's native final residue (Gln to Asn) accelerated activity such that it proceeded in vivo, while mutating the penultimate native His to Ser slowed splicing activity. We are currently studying the influence of high pressure on the activity of the inteins.Support or Funding InformationThis material is based upon work supported by the National Science Foundation under grants MCB‐0950245, MCB‐1244089 and MCB‐1517138 and by a Henry Dreyfus Teacher‐Scholar Award (KVM).