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