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

2. Industrial enzymes EnzymeSourceApplication Market(US$m) alkaline proteasesBacillusdetergents150 neutral proteasesBacillus, Aspergillusbaking, brewing 70 rennet proteasescalf, fungicheese making 60 isomerasesStreptomyceshigh glucose syrups 45 amylasesBacillus, Aspergillusstarch conversion, brewing, baking100 pectinasesAspergillusfruit/wine processing 40 other carbohydrasesfood processing 10 lipasesfungidetergents, food processing 20 specialty enzymesbacteria, fungifine chemicals

3. Extremophile enzymes Micro-organismsEnzymeApplication thermophiles50-110 o Cproteasesdetergents amylases, isomeraseshigh glucose syrups xylanasespaper bleaching DNA-polymerasesgenetic engineering psychrophiles5-20 o Cproteasescheese making proteases, lipasesdetergents alkaliphilespH>9proteases, lipasesdetergents

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

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

6. Subtilases: examples BACTERIA gram-positiveBacillussubtilisin, many others Thermoactinomycesthermitase Lactococcusnisin leader peptidase (NisP) cell-envelope proteinase (PrtP) gram-negative Thermusaqualysin Pseudomonasserine protease Pasteurellaserotype-specific antigen cyanobacteriaAnabaenaCa-dependent protease ARCHAEA halophilesHaloferaxhalolysin thermophilesPyrococcuspyrolysin Thermococcusstetterlysin

7. Subtilases: examples EUCARYA (lower) fungiTritirachiumproteinase K Aspergillusalkaline proteases yeastsSaccharomyceskexin, protease B, etc slime moldsDictyosteliumserine protease EUCARYA (higher) plantsArabidopsisserine proteases jellyfishHydrakexin-like protease nematodesCaenorhabditisblisterase tripeptidyl-peptidase insectsDrosophilafurin 1 and 2 molluscsAplysiafurin, PC1, PC2 amphibiaXenopusfurin, PC2 fishBranchiostomaPC1, PC2 mammalsHomo sapiensfurin, various PC’s

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

9. Homology modelling steps StepsTools Select protein sequence and familyBLAST, Pfam Select known X-ray structuresPDB Sequence alignmentFASTA, Clustal Create homology model frameworkQuanta Introduce deletions, insertionsQuanta, PDB Transfer new side chains to modelQuanta, Charmm Introduce S-S, ion binding sitesQuanta, Charmm Energy minimization (constraints !)Charmm Evaluate modelProCheck

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

11. Sequence alignment, molecular modelling (sub)families conserved residues large deletions and insertions Ca-binding sites S-S bonds substrate specificity Predictions (catalytic domain only):

12. Sequence alignment

13. 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

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

15. 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, Ca 2+ 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)

16. 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.