The conserved N-terminal helix of acylpeptide hydrolase from archeaon Aeropyrum pernix K1 is important for its hyperthermophilic activity.

The acylpeptide hydrolases from hyperthermophilic archaeon Aeropyrum pernix K1 has a short conserved N-terminal helix in its family. The role of this N-terminal helix in the function of the hyperthermophilic enzyme, however, is unknown. Here, we investigated this question by protein engineering and biophysical methods. We found that a mutant (DeltaN21) with the N-terminal helix deleted is no longer functional at the optimum temperature for WT enzyme (95 degrees C), required for the survival of Aeropyrum pernix K1. Instead, DeltaN21 has the optimum activity at approximately 77 degrees C, with higher activities than the WT enzyme below this temperature. DeltaN21 is less stable than the WT enzyme and started unfolding at approximately 77 degrees C, indicating that the loss of the enzymatic activity of DeltaN21 at higher temperature is due to its low thermodynamic stability. In addition, we found that the salt bridges formed between the N-terminal helix and the catalytic domain of the enzyme play only a minor role in stabilizing the enzyme, suggesting that hydrophobic interactions mainly contribute to the stabilization. Since the N-terminal helix is conserved in this family of enzymes, our results suggest that the N-terminal helix is likely to play an important role for stabilizing all other enzymes in this family.