Search

Abstract: 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 (ΔN21) with the N-terminal helix deleted is no longer functional at the optimum temperature for WT enzyme (95 °C), required for the survival of Aeropyrum pernix K1. Instead, ΔN21 has the optimum activity at ∼77 °C, with higher activities than the WT enzyme below this temperature. ΔN21 is less stable than the WT enzyme and started unfolding at ∼77 °C, indicating that the loss of the enzymatic activity of ΔN21 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. [Copyright &y& Elsevier]

Zearalenone hydrolase (ZHD) is the only reported α/β-hydrolase that can detoxify zearalenone (ZEN). ZHD has demonstrated its potential as a treatment for ZEN contamination that will not result in damage to cereal crops. Recent researches have shown that the V153H mutant ZHD increased the specific activity against α-ZOL, but decreased its specific activity to β-ZOL. To understand whyV153H mutation showed catalytic specificity for α-ZOL, four molecular dynamics simulations combining with protein network analysis for wild type ZHD α-ZOL, ZHD β-ZOL, V153H α-ZOL, and V153H β-ZOL complexes were performed using Gromacs software. Our theoretical results indicated that the V153H mutant could cause a conformational switch at the cap domain (residues Gly161–Thr190) to affect the relative position catalytic residue (H242). Protein network analysis illustrated that the V153H mutation enhanced the communication with the whole protein and residues with high betweenness in the four complexes, which were primarily assembled in the cap domain and residues Met241 to Tyr245 regions. In addition, the existence of α-ZOL binding to V153H mutation enlarged the distance from the OAE atom in α-ZOL to the NE2 atom in His242, which prompted the side chain of H242 to the position with catalytic activity, thereby increasing the activity of V153H on the α-ZOL. Furthermore, α-ZOL could easily form a right attack angle and attack distance in the ZHD and α-ZOL complex to guarantee catalytic reaction. The alanine scanning results indicated that modifications of the residues in the cap domain produced significant changes in the binding affinity for α-ZOL and β-ZOL. Our results may provide useful theoretical evidence for the mechanism underlying the catalytic specificity of ZHD. [ABSTRACT FROM AUTHOR]

Abstract: Acylpeptide hydrolase of Aeropyrum pernix K1 is composed of a catalytic α/β hydrolase domain and a non-catalytic β-propeller domain. The Glu88 residue of the propeller domain is highly conserved in the prolyl oligopeptidase family and forms an inter-domain salt bridge with Arg526, a key residue for substrate binding. We have dissected the functions of Glu88 using site-directed mutagenesis, steady-state kinetics analyses, and molecular dynamics simulations. In E88A and E88A/R526K mutants, with a broken inter-domain salt bridge and a positive charge at position 526, catalytic activities for both a peptidase substrate and an esterase substrate were almost abolished. Analysis of the pH dependence of the mutants'' reaction kinetics indicates that these mutations lead to changes in the electrostatic environment of the active site, which can be modulated by chloride ions. These findings indicate that the neutralization at position 526 is favorable for the activity of the enzyme, which is also verified by the catalytic behavior of E88A/R526V mutant. All mutants have lower thermodynamic stability than the wild-type. Therefore, Glu88 plays two major roles in the function of the enzyme: neutralizing the positive charge of Arg526, thereby increasing the enzymatic activity, and forming the Glu88–Arg526 salt bridge, thereby stabilizing the protein. [Copyright &y& Elsevier]