Molecular Basis Of Thermal Adaptation Of Pyrolysin From Hyperthermophilic Archaeon Pyrococcus Furiosus

The marine hyperthermophilic archaeon Pyrococcus furiosus which grows optimally at100?was isolated from geothermally heated marine sediments at the beach of Porto di Levante, Vulcano, Italy. The organism is highly proteolytic and requires peptides for growth. Pyrolysin is an extracellular serine protease that was isolated from the cell envelope fraction of P. furiosus. This enzyme was characterized as an extremely thermostable protease with optimal caseinolytic activity at115?and a half-life of4h at100?. As one of the most thermostable enzymes, pyrolysin is an ideal model for investigating the molecular basis of enzymatic activity and stability near the maximum temperature of life.Pyrolysin is grouped into the pyrolysin family of subtilase, the precursor of pyrolysin contains a class ? signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain and a C-terminal extension. As an extracellular protease, pyrolysin needs to perform its maturation process and fulfill its extracellular function at high temperatures in seawater containing metal ions. But litter is known about the effects of metal ions on the properties of pyrolysin. Besides, pyrolysin is a cell-envelope associated protease, and S-layer protein(s) is the main component in the cell envelope of P. furiosus, whether S-layer protein(s) exerts effects on the properties of pyrolysin is underdetermined. In this study, the recombinant pro form of pyrolysin expressed in Escherichia coli is used to analyze the effects of metal ions and S-layer protein on properties of pyrolysin, in order to gain insights into its potential stabilizing mechanism and further modification for stability improvement. Firstly, we investigate the effects of the main metal ions in seawater (Na+?Ca2+?Mg2+) on maturation and enzyme properties of pyrolysin. Our results suggest that thesupplementation of Na+, Ca2+or Mg2+salts of similar concentrations to those of thesemetal ions in seawater destabilizes the recombinant pyrolysin but leads to an increasein enzyme activity. According to the molecular architecture of pyrolysin, thedestabilizing effect of metal ions on pyrolysin appears to be related to the disturbanceof surface electrostatic interactions of the enzyme. Secondly, several residues that were predicted to be involved in Ca2+-binding inpyrolysin were subjected to mutational analysis. Based on the biochemicallycharacterization of these amino acid substitution mutants, we identified twohigh-affinity Ca2+-binding sites (Cal and Ca2) which are separately located incatalytic domain and C-terminal extension. These experiments reveal that the bindingof Ca2+is important for thermostability of pyrolysin. Interestingly, the Asnsubstitutions at the residues Asp818and Asp820of the Ca2site result in theimprovement of both enzyme thermostability and activity without affecting itsCa2+-binding affinity, most likely due to the elimination of unfavorable electrostaticrepulsion in the Ca2site. Given the effects of metal ions (Na+?Ca2+?Mg2+) on theproperties of pyrolysin, all these results suggest that metal ions in seawater playimportant roles in modulating the stability and activity of pyrolysin. In addition, theresults also show that proper manipulation of electrostatic interactions appears to bean effective strategy for further stabilization of hyperthermophilic enzymes, and thisoffers new molecular strategies for probing the maximum temperature whichprotein/protease could withstand. Finally, the recombinant S-layer protein of P. furiosus, S1(PF1399), wassuccessfully expressed in E. coli. And we use the recombinant S1to investigate theeffects of S1on the maturation and stability of pyrolysin. Our results suggest that S1improves the maturation efficiency of pyrolysin, mainly through the increase in theproper folding efficiency of the proform Pls. In addition, S1can stabilize the maturepyrolysin in the presence of high concentrations of Na+. Our results may provideimportant clues about the role of S-layer protein S1in the adaptation of pyrolysin tothe hyperthermal marine environment.