Extein residues regulate the catalytic function of Spl DnaX intein enzyme by restricting the near-attack conformations of the active-site residues.

Abstract

Intein enzymes catalyze the splicing of their flanking polypeptide chains and have found tremendous biotechnological applications. Their terminal residues form the catalytic core and participate in the splicing reaction. Hence, the neighboring N- and C-terminal extein residues influence the catalytic rate. As these extein residues vary depending on the substrate identity, we tested the influence of 20 amino acids at these sites in the Spl DnaX intein and observed significant variation of spliced product as well as N- and C-terminus cleavage product formation. We investigated the dependence of these reactions on the extein residues by molecular dynamics (MD) simulations on 8 extein variants, and found that the conformational sampling of the active-site residues of the intein enzyme differed among these extein variants. We found that the extein variants that sample higher population of near-attack conformers of the active-site residues undergo higher product formation in our activity assays. Ground state conformers that closely resemble the transition state are referred to as near-attack conformers (NACs). Very good correlation was observed between the NAC populations from the MD simulations of 8 extein variants and the corresponding product formation from our activity assays. Furthermore, this molecular detail enabled us to elucidate the mechanistic roles of several conserved active-site residues in the splicing reaction. Overall, this study shows that the catalytic power of Spl DnaX intein enzyme, and most likely other inteins, depends on the efficiency of formation of near-attack conformers in the ground state, which is further modulated by the extein residues. This article is protected by copyright. All rights reserved.