Transcript Document

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
Glucokinases
Hexokinases
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
Hexokinases
Glucokinases
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
Protein engineering
Super-family
WT = P27Y28 Y28G3DM:
-> 10%
Y28GP27A
-> 200%platforms
control P27A -> 15%
Y28GP27E -> 85%
Y28GP27R -> 80%
G28 -> P,A -> no act.
3DM: Protein engineering
Super-family
Y28DP27G
-> 300%platforms
Y28RP27N
all others less active as wild type
-> 200%
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
Inhibitor Design
OH
ICL:
O-
O- OH
O-
HO
HO
O
Glu(100%)
O
-
OH
O-
OAH:
OH
HO
O-
HO
O-
OH
O-
OH
OH
3DM: Protein engineering Super-family platforms
3DM: Protein engineering Super-family platforms
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
3DM: Protein engineering Super-family platforms
Inhibitor Design
HO
O-
OH
O-
F
OH
OH
O-
OH
O-
OH
F
100% Diol
3DM: Protein engineering Super-family platforms
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!!!!!
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
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.
3DM: Protein engineering Super-family platforms
Thank you for your attention