Functional Analysis Of Inserts In Pyrolysin From Hyperthermophilic Pyrococcus Furiosus And Autocleavage Mechanism Of The Enzyme

Isolated from the cell envelope fraction of hyperthermophilic marine archaeon Pyrococcus furiosus,pyrolysin is the prototype of the pyrolysin family of the subtilisin-like serine protease superfamily(subtilases).Members of pyrolysin family are characterized by large inserts and/or long C-terminal extensions.Pyrolysin is an extremely stable enzyme with a half-life of 4 h at 100? and maximum activity at 115?.Taking advantage of its hyperthermostability,pyrolysin is an ideal model for investigating the molecular basis of thermal adaptation of proteins.Pyrolysin is synthesized as a precursor containing a signal peptide,an N-terminal propeptide,a subtilisin-like catalytic domain and a C-terminal extension(CTE).This enzyme experiences autocatalytic activation at high temperatures to turn into the mature form by autoprocessing of its N-and C-terminal propeptides.The CTE of pyrolysin is important for enzyme stability and activity,but the functional roles of the unique inserts in the catalytic domain of pyrolysin are still unknown.As an extracellular protease of P.furiosus,pyrolysin functions at high temperatures in seawater containing metal ions.Unexpectedly,we previously found that pyrolysin suffered autocleavage and precipitation when incubated with metal ions.In this study,we have investigated the mechanisms of pyrolysin autocleavage and precipitation,in order to gain insights into the molecular basis of adaptation of this enzyme to hyperthermal marine environments.Pyrolysin contains four unique inserts(Is 147,Is29,Is27,and Is8)in the catalytic domain.To investigate the functions of these inserts,we performed domain deletions and found that three inserts are either essential(Is 147 and Is27)or important(Is8)for efficient maturation of pyrolysin.The large insert Is 147 contains two novel Ca2+-binding sites(Ca3 and Ca4)consisted of two calcium-binding Dx[DN]xDG motifs that are conserved in many pyrolysin-like proteases.Mutagenesis revealed that the Ca3 site contributes to enzyme thermostability and the Ca4 site is necessary for pyrolysin to fold into a maturation-competent conformation.Mature insert-deletion variants were characterized and showed that Is29 and Is8 contribute to enzyme activity and stability,respectively.In the presence of metal ions,pyrolysin undergoes autocleavage in an intermolecular manner to generate five fragments.Two specific autocleavage sites have been identified by the N-terminal sequencing and both of them are located within the inserts(Is29 and Is27).Mutagenesis revealed that disruption of the ion pairs in Is27 and Is8 induces autocleavage of pyrolysin in the absence of salts.Although pyrolysin suffered autocleavage in the presence of metal ions,the autocleavage products combine noncovalently to form an active,nicked enzyme.In addition,a Glu substitution at residue Arg249 in Is29 increases the resistance to salt-induced autocleavage and the thermostability of this enzyme.This result indicates that modification of extra structure elements of pyrolysin-like proteases is a promising strategy to further stabilize this class of enzyme.Both of the proform and mature form of pyrolysin tends to precipitate in the presence of metal ions.In this study,transmission electron microscopy(TEM)was employed to characterize the morphological features of the aggregates formed by pyrolysin in artificial seawater.We found that pyrolysin proform formed amorphous aggregates,while the mature enzyme formed amyloid-like fibrils.Interestingly,protein substrates can regulate the aggregate process of the mature enzyme.On one hand,protein substrates can prevent the mature enzyme from aggregation.On the other hand,protein substrates can make the aggregated mature enzyme revert to soluble form.Our results suggest that the protein substrates can not only inhibit the autocleavage of pyrolysin,but also keep the enzyme soluble in the hyperthermal marine environment.