find the best starting enzyme find the hotspots select the best mutations at these hotspots
DESCRIPTION
Find the best starting enzyme Find the hotspots Select the best mutations at these hotspots. Glucokinases. Hexokinases. Allowed amino acids at pos. 64 (46 in alignment) = A,G,P,S,C. Hexokinases. Allowed amino acids: A,G,P,S. - PowerPoint PPT PresentationTRANSCRIPT
3DM: Protein engineering Super-family platforms
1. Find the best starting enzyme
2. Find the hotspots
3. Select the best mutations at these hotspots
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
Hexokinases
Glucokinases
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
Allowed amino acids at pos. 64 (46 in alignment) = A,G,P,S,C
3DM: Protein engineering Super-family platforms
HexokinasesGlucokinases
Allowed amino acids:
A,G,P,S
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
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.
3DM: Protein engineering Super-family platforms
Cupin superfamily
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platformsWT = P27Y28 Y28GP27A -> 200% control P27A -> 15% Y28GP27E -> 85% Y28GP27R -> 80%
Y28G -> 10%
G28 -> P,A -> no act.
3DM: Protein engineering Super-family platformsY28DP27G -> 300% Y28RP27N -> 200% all others less active as wild type
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
3DM: Protein engineering Super-family platforms
ICL:OH
O-
OHHO
O-
HOO-
O O-
OH
HOO-
OAH:OH O-
OH
HO
O- OHOH
O- Glu(100%)
Inhibitor Design
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
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 wildtypeOAH:
Ser157Pro: Changed specificity. Increased the affinity of OAH for a isocitrate like compound
Petal death protein:
Ser157Pro: Selectively removed OAH activity
Inhibitor Design
3DM: Protein engineering Super-family platforms
OH O-
F
F
O- OH
Inhibitor Design
OH O-
OH
HO
O- OH
100% Diol
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
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
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!!!!
3DM: Protein engineering Super-family platforms
Thank you for your attention