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Hyperthermophile subtilases

Hyperthermophile subtilases. Homology modelling of two subtilisin-like serine proteases from the hyperthermophile archaea Pyrococcus furiosus and Thermococcus stetteri Protein Engineering 10 (1997) 905-914. Wilfried Voorhorst, Angela Warner, Willem de Vos

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Hyperthermophile subtilases

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  1. Hyperthermophile subtilases Homology modelling of two subtilisin-like serine proteases from the hyperthermophile archaea Pyrococcus furiosus and Thermococcus stetteri Protein Engineering 10 (1997) 905-914 Wilfried Voorhorst, Angela Warner, Willem de Vos Wageningen University, Wageningen, the Netherlands Roland J. Siezen NIZO food reseach, Ede, the Netherlands

  2. Temperature-dependent subtilases temp.optimum protease structure psychrophile Bacillus TA41 0 - 5 oC subtilisinTA41 model mesophile Bacillus amyloliquefaciens 40 - 50 oC subtilisinBPN’ X-ray thermophile Thermoactinomyces vulgaris 60 - 70 oC thermitase X-ray hyperthermophile Thermococcus stetteri 80 - 90 oC stetterlysin model Pyrococcus furiosus 95 -100 oC pyrolysin model

  3. Protein stabilization • H-bonds • S-S bonds • hydrophobic interactions • aromatic interactions • salt bridges (ion pairing) • helix dipole, helix capping • shorter loops • glycosylation • In general: REDUCE FLEXIBILITY !!

  4. Sequence alignment

  5. Modelling of insertions, deletions +147 +29 +27 +8 +6 +5 +4 -2 +2 +6 • Based on sequence alignment: • introduce deletions • add insertions (< 7 residues) Pyrolysin model = modelled = not modelled

  6. Pyrolysin: charged residues red: acidic blue: basic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

  7. Pyrolysin: aromatic residues purple: aromatic yellow: inserts ftp://ftp.cmbi.kun.nl/pub/molbio/siezen97/

  8. Catalytic domain properties

  9. General conclusions • Homology model is only a first approximation: • several (large) loops cannot be modeled • interaction of surface residues difficult to model (no H2O) • BUT: model suffices for general predictions • Thermostability of hyperthermophilic enzymes may be correlated with: • increase of ionic interactions, networks • increase of aromatic interactions, clusters

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