Protein Interface and

Active Site Redesign

Ed Triplett

SBB251/CS260/CBB230

Algorithms in Structural Molecular Biology and Biophysics

03/27/08

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.

Enzymatic Promiscuity

• Substrate - with same reaction

• Catalytic - different substrates and reactions

• Product - same substrate, similar reaction to produce

new product

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.

Minimal Redesign

• Relies on intact scaffold of w.t. enzyme.

• Rarely approaches efficiency of natural enzymes

• May point the way for future design work.

Catalytic Triad

http://herkules.oulu.fi/isbn9514268385/html/graphic77.png

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.

Subtilisin Derivatives

Hilvert et al, 2007.

Subtilisin vs. Selenosubtilisin

Hilvert et al, 2007.

Interconverting Homologous Enzymes

• AEE and MLE2 -> OSBS

• 3-alpha-HSD and 5-beta reductase

• MUTY

• HisA/F ->TrpF

OSBS activity in AEE and MLE2

Hilvert et al, 2007.

• AEE =

light blue

• MLE II = yellow

• OSBS = green

Hilvert et al, 2007.

Introduction of Catalytic Machinery

• Uses selectivity of original for substrates

• STYX

• Cyproase

Cyproase

Quemeneur et al, 1998.

Removing Cat. Nucleophiles

• Reveals previously blocked reaction pathways.

• Gluconate-CoA transferase

• GAPDH

• Glycosidases

Hilvert et al, 2007.

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).

Farnesyl diphosphate polymerase

Hilvert et al, 2007.

Product Promiscuity

Hilvert et al, 2007.

Improving Promiscuity

• GST quintuple mutant

• Cofactor promiscuity - PLP

Hilvert et al, 2007.

Hilvert et al, 2007.

Exploiting Dunathan’s Hypothesis to convert alanine

racemase into an aldolase

Directed Evolution

• Can rescue dysfunctional domain interfaces in chimeric NRPSs.

Fischbach et al, 2007.

Sources

Angew Chem Int Ed Engl. 2007;46(18):3212-36. Minimalist active-site redesign: teaching old enzymes new tricks. Toscano MD, Woycechowsky KJ, Hilvert D.

Proc Natl Acad Sci U S A. 2007 Jul 17;104(29):11951-6. 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: 301-304. Engineering cyclophilin into a proline-specific endopeptidase. Quemeneur E, Moutiez M, Charbonnier J-B, Menez A.