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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.
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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.