1. 3DM: Protein engineering Super-family platforms 1.Find the best starting enzyme 2.Find the hotspots 3.Select the best mutations at these hotspots

2. 3DM: Protein engineering Super-family platforms

4. Hexokinases Glucokinases

5. 3DM: Protein engineering Super-family platforms

11. Allowed amino acids at pos. 64 (46 in alignment) = A,G,P,S,C

12. 3DM: Protein engineering Super-family platforms Hexokinases Glucokinases Allowed amino acids: A,G,P,S

13. 3DM: Protein engineering Super-family platforms

14. Can we change a hexokinase into a glucokinase? With a smart library (size 32) targeted at these correlating positions -> 10 fold increase of glycokinase activity.

15. 3DM: Protein engineering Super-family platforms Cupin superfamily

16. 3DM: Protein engineering Super-family platforms

17. WT = P27Y28 Y28GP27A -> 200% control P27A -> 15% Y28GP27E -> 85% Y28GP27R -> 80% Y28G -> 10% G28 -> P,A -> no act.

18. 3DM: Protein engineering Super-family platforms Y28DP27G -> 300% Y28RP27N -> 200% all others less active as wild type

19. 3DM: Protein engineering Super-family platforms Inhibitor Design The ICL-like superfamily: * OAH is a member * Used by fungi produce oxalate (toxic) The most conserved residues ( >97% ) Correlating positions

20. 3DM: Protein engineering Super-family platforms ICL: OH O-O- HO O-O- O-O- O O-O- OH HO O-O- OAH: OH O-O- HO O-O- OH O- Glu(100%) Inhibitor Design

21. 3DM: Protein engineering Super-family platforms

22. OAH: Ser157 -> A, P or T : Km = 1200, 600, 800 x WT kcat = 0.2, 0.3, 0.4 X WT ICL: Pro157 -> S: Km = 50 x WT, kcat = equal to wildtype OAH: Ser157Pro: Changed specificity. Increased the affinity of OAH for a isocitrate like compound Petal death protein: Ser157Pro: Selectively removed OAH activity Inhibitor Design

23. 3DM: Protein engineering Super-family platforms OH O-O- F F O-O- Inhibitor Design OH O-O- HO O-O- OH 100% Diol

24. 3DM: Protein engineering Super-family platforms Correlating different data types: Correlation between fungi that are known oxalic acid producers and the amino acids in the alignment. -> make subset Select all amino acids that are conserved within this group and a different residue in the rest of the alignment. The best scoring amino acid = S157!!!!! Subsets and data comparison

25. 3DM: Protein engineering Super-family platforms

29. Enantioselectivity: Select all mutations from articles that are known to have effect on enantioselectivity and plot these in the protein of interest. Design library at these positions taking high # of positions with low # of different aa. Thermostability: Select positions that are flexible in the structure (B-FIT values/RMSD) excluding parts that move to perform the function Introduce the most common residues at these positions Smart library design Selectivity: Select mutations from articles that are known to have effect on specificity and/or use correlated mutation data. Always think!!!!

30. 3DM: Protein engineering Super-family platforms Thank you for your attention