title: Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri.
abstract: The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases 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 modelling of known substrates in the substrate-binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.
authors.affiliation: Department of Microbiology, Wageningen Agricultural University, The Netherlands.
authors.forename: W G
authors.initials: WG
authors.lastname: Voorhorst
authors.forename: A
authors.initials: A
authors.lastname: Warner
authors.forename: W M
authors.initials: WM
authors.lastname: de Vos
authors.forename: R J
authors.initials: RJ
authors.lastname: Siezen
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry; D019843:Archaeal Proteins; D001665:Binding Sites; D003001:Cloning, Molecular; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry; D019712:Thermococcus / Q000201:enzymology* / Q000235:genetics
chemical_list: D000596:Amino Acids; D019843:Archaeal Proteins; D012697:Serine Endopeptidases; D013381:Subtilisins; C101133:pls protein, Pyrococcus furiosus; C110156:stetterlysin