Protein Interface and Active Site Redesign Ed Triplett SBB251/CS260/CBB230 Algorithms in Structural...
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Transcript of Protein Interface and Active Site Redesign Ed Triplett SBB251/CS260/CBB230 Algorithms in Structural...
![Page 1: Protein Interface and Active Site Redesign Ed Triplett SBB251/CS260/CBB230 Algorithms in Structural Molecular Biology and Biophysics 03/27/08.](https://reader036.fdocuments.us/reader036/viewer/2022062517/56649f1c5503460f94c32d80/html5/thumbnails/1.jpg)
Protein Interface and
Active Site Redesign
Ed Triplett
SBB251/CS260/CBB230
Algorithms in Structural Molecular Biology and Biophysics
03/27/08
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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.
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Enzymatic Promiscuity
• Substrate - with same reaction
• Catalytic - different substrates and reactions
• Product - same substrate, similar reaction to produce
new product
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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.
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Minimal Redesign
• Relies on intact scaffold of w.t. enzyme.
• Rarely approaches efficiency of natural enzymes
• May point the way for future design work.
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Catalytic Triad
http://herkules.oulu.fi/isbn9514268385/html/graphic77.png
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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.
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Subtilisin Derivatives
Hilvert et al, 2007.
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Subtilisin vs. Selenosubtilisin
Hilvert et al, 2007.
![Page 10: Protein Interface and Active Site Redesign Ed Triplett SBB251/CS260/CBB230 Algorithms in Structural Molecular Biology and Biophysics 03/27/08.](https://reader036.fdocuments.us/reader036/viewer/2022062517/56649f1c5503460f94c32d80/html5/thumbnails/10.jpg)
Interconverting Homologous Enzymes
• AEE and MLE2 -> OSBS
• 3-alpha-HSD and 5-beta reductase
• MUTY
• HisA/F ->TrpF
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OSBS activity in AEE and MLE2
Hilvert et al, 2007.
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• AEE =
light blue
• MLE II = yellow
• OSBS = green
Hilvert et al, 2007.
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Introduction of Catalytic Machinery
• Uses selectivity of original for substrates
• STYX
• Cyproase
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Cyproase
Quemeneur et al, 1998.
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Removing Cat. Nucleophiles
• Reveals previously blocked reaction pathways.
• Gluconate-CoA transferase
• GAPDH
• Glycosidases
Hilvert et al, 2007.
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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).
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Farnesyl diphosphate polymerase
Hilvert et al, 2007.
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Product Promiscuity
Hilvert et al, 2007.
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Improving Promiscuity
• GST quintuple mutant
• Cofactor promiscuity - PLP
Hilvert et al, 2007.
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Hilvert et al, 2007.
Exploiting Dunathan’s Hypothesis to convert alanine
racemase into an aldolase
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Directed Evolution
• Can rescue dysfunctional domain interfaces in chimeric NRPSs.
Fischbach et al, 2007.
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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.