Find the best starting enzyme

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

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Allowed amino acids at pos. 64 (46 in alignment)

A,G,P,S,C
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Can we change a hexokinase into a
glucokinase? With a smart library (size 32)
targeted at these correlating positions -gt 10
fold increase of glycokinase activity.
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Cupin superfamily
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WT P27Y28
Y28GP27A -gt 200 control P27A -gt 15 Y28GP27E
-gt 85 Y28GP27R -gt 80
G28 -gt P,A -gt no act.
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Y28DP27G -gt 300 Y28RP27N -gt 200 all
others less active as wild type
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Inhibitor Design
The ICL-like superfamily OAH is a member
Used by fungi produce oxalate (toxic)
The most conserved residues ( gt97 )
Correlating positions
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Inhibitor Design
ICL
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Inhibitor Design
OAH Ser157 -gt A, P or T Km 1200, 600, 800
x WT kcat 0.2, 0.3, 0.4 X WT
ICL Pro157 -gt 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
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Inhibitor Design
OH
O-
OH
F
O-
HO
F
O-
OH
OH
O-
OH
100 Diol
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Subsets and data comparison
Correlating different data types Correlation
between fungi that are known oxalic acid
producers and the amino acids in the alignment.
-gt 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!!!!!
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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.
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Thank you for your attention