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Homology modelling of serine proteases

Homology modelling of serine proteases. Industrial examples. Roland J. Siezen. Industrial enzymes. Enzyme Source Application Market(US$m) alkaline proteases Bacillus detergents 150 neutral proteases Bacillus, Aspergillus baking, brewing 70

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Homology modelling of serine proteases

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  1. Homology modelling of serine proteases Industrial examples Roland J. Siezen

  2. Industrial enzymes Enzyme Source Application Market(US$m) alkaline proteases Bacillus detergents 150 neutral proteases Bacillus, Aspergillus baking, brewing 70 rennet proteases calf, fungi cheese making 60 isomerases Streptomyces high glucose syrups 45 amylases Bacillus, Aspergillus starch conversion, brewing, baking 100 pectinases Aspergillus fruit/wine processing 40 other carbohydrases food processing 10 lipases fungi detergents, food processing 20 specialty enzymes bacteria, fungi fine chemicals

  3. Extremophile enzymes Micro-organisms Enzyme Application thermophiles 50-110oC proteases detergents amylases, isomerases high glucose syrups xylanases paper bleaching DNA-polymerases genetic engineering psychrophiles 5-20oC proteases cheese making proteases, lipases detergents alkaliphiles pH>9 proteases, lipases detergents

  4. Subtilases:the superfamily of subtilisin-like serine proteases Protein Science 6 (1997) 501-523 Roland J. Siezen NIZO food reseach, Ede, the Netherlands Jack A.M. Leunissen CMBI, Nijmegen, the Netherlands

  5. Subtilases: properties catalytic triad Asp - His - Ser pre-pro-proteases autocatalytic maturation over 200 amino acid sequences few X-ray structures (a/b type) S H D pre pro catalytic other

  6. Subtilases: examples BACTERIA gram-positive Bacillus subtilisin, many others Thermoactinomyces thermitase Lactococcus nisin leader peptidase (NisP) cell-envelope proteinase (PrtP) gram-negative Thermus aqualysin Pseudomonas serine protease Pasteurella serotype-specific antigen cyanobacteria Anabaena Ca-dependent protease ARCHAEA halophiles Haloferax halolysin thermophiles Pyrococcuspyrolysin Thermococcus stetterlysin

  7. Subtilases: examples EUCARYA (lower) fungi Tritirachium proteinase K Aspergillus alkaline proteases yeasts Saccharomyces kexin, protease B, etc slime molds Dictyostelium serine protease EUCARYA (higher) plants Arabidopsis serine proteases jellyfish Hydra kexin-like protease nematodes Caenorhabditis blisterase tripeptidyl-peptidase insects Drosophila furin 1 and 2 molluscs Aplysia furin, PC1, PC2 amphibia Xenopus furin, PC2 fish Branchiostoma PC1, PC2 mammals Homo sapiens furin, various PC’s

  8. Subtilase: structure catalytic domain C ~ 300 residues / sec.structure N Ser Asp His Ca1 substrate binding cleft Ca2

  9. Homology modelling steps • Steps Tools • Select protein sequence and family BLAST, Pfam • Select known X-ray structures PDB • Sequence alignment FASTA, Clustal • Create homology model framework Quanta • Introduce deletions, insertions Quanta, PDB • Transfer new side chains to model Quanta, Charmm • Introduce S-S, ion binding sites Quanta, Charmm • Energy minimization (constraints !) Charmm • Evaluate model ProCheck

  10. X-ray structures subtilisin BPN’: thick:red, yellow thermitase: thin:green, blue 3-D alignment superimpose identical residues  conserved core of -helices and -sheet strands

  11. Sequence alignment

  12. Create homology model framework • Based on sequence alignment: • select the segments of • subtilisin BPN’ (green) and • thermitase (red) with • highest sequence identity • best loop length

  13. 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

  14. Completing the model • Transfer new side chains to model • new amino acid sequence • regularize molecule (Quanta/Charmm) • Introduce S-S, ion binding sites • S-S bonds, Ca2+ binding sites • Energy minimization • constrain catalytic residues (Asp, His, Ser, Asn) • constrain substrate binding region (-sheet strands) • energy minimize (Charmm) • Evaluate model parameters • Ramachandran plot: main chain phi-psi angles • side chain parameters (ProCheck)

  15. Conclusions Very extensive modification of the basic “subtilisin”structure is allowed. Stability and proteolytic specificity can be dramatically altered. NATURE has engineered subtilases beyond our wildest dreams and provided lessons and rules to guide us in protein engineering in general.

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