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Enzyme Optimization for Glycokinase Activity Enhancement

Explore how to improve glycokinase activity through smart library design and targeted mutations at specific hotspots. Investigate the conversion of hexokinase to glucokinase using allowed amino acids and Cupin superfamily mutations. Discover optimal inhibitors based on correlating data from ICL-like superfamily and OAH. Utilize enantioselectivity, thermostability, and selectivity mutations for enhanced enzyme performance.

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Enzyme Optimization for Glycokinase Activity Enhancement

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  1. Find the best starting enzyme • Find the hotspots • Select the best mutations at these hotspots

  2. Glucokinases Hexokinases

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

  4. Hexokinases Allowed amino acids: A,G,P,S Glucokinases

  5. 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.

  6. Cupin superfamily

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

  8. Y28DP27G -> 300% Y28RP27N -> 200% all others less active as wild type

  9. Inhibitor Design The ICL-like superfamily: * OAH is a member * Used by fungi produce oxalate (toxic) The most conserved residues ( >97% ) Correlating positions

  10. O O- Glu(100%) OAH: O- OH OH HO O- O- OH O- OH O- HO HO OH OH HO O- O- OH Inhibitor Design ICL:

  11. Inhibitor Design 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

  12. Inhibitor Design OH OH O- F O- HO F O- OH OH O- OH 100% Diol

  13. Subsets and data comparison 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!!!!!

  14. Smart library design Always think!!!! • 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 • Selectivity: • Select mutations from articles that are known to have effect on specificity and/or use correlated mutation data.

  15. Thank you for your attention

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