Protein Interface and Active Site Redesign

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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.

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

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.