1. Protein Interface and Active Site Redesign
Ed Triplett
SBB251/CS260/CBB230
Algorithms in Structural Molecular Biology and Biophysics
03/27/08

2. Minimal Active Site Redesign
Enzymes are fine-tuned for the reactions they catalyze, to present an optimized environment for catalysis.
Redesign requires undoing some of this customization and the specificity it offers.
Focus on homology and promiscuity.

3. Enzymatic Promiscuity
Substrate - with same reaction
Catalytic - different substrates and reactions
Product - same substrate, similar reaction to produce new product

4. Enzyme Homology
Enzymes are very tolerant to mutations in residue positions distant from the active site.
Proteins that fold into remarkably similar structures may show little sequence homology.

5. Minimal Redesign Relies on intact scaffold of w.t. enzyme.
Rarely approaches efficiency of natural enzymes
May point the way for future design work.

6. Catalytic Triad

7. Subtilisin
Well studied serine protease, containing the famed Ser-His-Asp catalytic triad.
Replace -OH of serine with -SH to yield esterase with aminolysis activity.
S221C/P225A = subtiligase
Replace serine with selenocysteine to produce the peroxidase selenosubtilisin.

8. Subtilisin Derivatives
Hilvert et al, 2007.

9. Subtilisin vs. Selenosubtilisin
Hilvert et al, 2007.

10. Interconverting Homologous Enzymes
AEE and MLE2 -> OSBS
3-alpha-HSD and 5-beta reductase
MUTY
HisA/F ->TrpF

11. OSBS activity in AEE and MLE2
Hilvert et al, 2007.

12. AEE =
light blue
MLE II = yellow
OSBS = green
Hilvert et al, 2007.

13. Introduction of Catalytic Machinery
Uses selectivity of original for substrates
STYX
Cyproase

14. Cyproase
Quemeneur et al, 1998.

15. Removing Cat. Nucleophiles
Reveals previously blocked reaction pathways.
Gluconate-CoA transferase
GAPDH
Glycosidases
Hilvert et al, 2007.

16. Partitioning of Rxn Intermediates
hydrolase -> ligase : Who gets the acyl group?
Phosphoesterase rescue from suicide inhibitor.
Control over polymerization (pocket size) and stereoselectivity (orientation of substrate and catalytic residues).

17. Farnesyl diphosphate polymerase
Hilvert et al, 2007.

18. Product Promiscuity
Hilvert et al, 2007.

19. Improving Promiscuity
GST quintuple mutant
Cofactor promiscuity - PLP
Hilvert et al, 2007.

20. Exploiting Dunathan’s Hypothesis to convert alanine racemase into an aldolase
Hilvert et al, 2007.

21. Directed Evolution
Can rescue dysfunctional domain interfaces in chimeric NRPSs.
Fischbach et al, 2007.

22. Sources
Angew Chem Int Ed Engl. 2007;46(18): Minimalist active-site redesign: teaching old enzymes new tricks. Toscano MD, Woycechowsky KJ, Hilvert D.
Proc Natl Acad Sci U S A Jul 17;104(29): Epub 2007 Jul 9. Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes. Fischbach MA, Lai JR, Roche ED, Walsh CT, Liu DR.
Nature. 1998; 391: Engineering cyclophilin into a proline-specific endopeptidase. Quemeneur E, Moutiez M, Charbonnier J-B, Menez A.