HOMOLOGY MODELING OF 2 SUBTILISIN-LIKE SERINE PROTEASES FROM THE HYPERTHERMOPHILIC ARCHAEA PYROCOCCUS-FURIOSUS AND THERMOCOCCUS-STETTERI

The hyperthermophilic archaeon Pyrococcus furiosus produces an extrace llular, glycosylated hyperthermostable subtilisin-like serine protease , termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., P latteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 2 0426-20431), Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophil ic archaeon Thermococcus stetteri, Like pyrolysin, the deduced sequenc e of this serine protease, designated stetterlysin, contains a catalyt ic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures, Comparison of the pr edicted three-dimensional models of the catalytic domain of stetterlys in and pyrolysin with the crystal structure of subtilases from mesophi lic and thermophilic origin, i.e., subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led t o the identification of features that could be related to protein stab ilization, Higher thermostability was found to be correlated with an i ncreased number of residues involved in pairs and networks of charge-c harge and aromatic-aromatic interactions, These highly thermostable pr oteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues, The latter are often present in putative N-glycosylation sites, Results from modellin g of known substrates in the substrate-binding region support the broa d substrate range and the autocatalytic activation previously suggeste d for pyrolysin.