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Evolution of Highly Active Enzymes by Homology-Independent Recombination

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Evolution of Highly Active Enzymes by Homology-Independent ... alpha, beta, delta, zeta, theta, mu, pi, sigma, tau, phi, omega. G-site (GSH-binding domain) ... – PowerPoint PPT presentation

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Title: Evolution of Highly Active Enzymes by Homology-Independent Recombination


1
Evolution of Highly Active Enzymes by
Homology-Independent Recombination
Griswold, K. E. Kawarasaki, Y. Ghoneim, N.
Benkovic, S. J. Iverson, B. L. Georgiou, G.
Proc. Natl. Acad. Sci. USA 2005, 102, 10082-10087.
Lisa Cooper Biochemistry September 7, 2005
2
Outline
  • Background
  • Recombination methods
  • Combinatorial generation of protein chimeras
  • Theta-class GST enzymes
  • Experimental Results
  • Flow cytometry screening
  • Enzyme characterization
  • Conclusions
  • Potential roles for recombination methods
  • Ongoing and future research

3
Techniques
  • Homology-dependent methods
  • DNA family shuffling
  • Homology-independent methods
  • ITCHY
  • SCRATCHY
  • PCR methods
  • Recombination-dependent exponential amplification
  • Overlap extension
  • Reassembly

4
The ITCHY and SCRATCHY Show !!!
5
ITCHY
  • Incremental Truncation for the Creation of Hybrid
    Enzymes
  • More diverse set of functional fusions than DNA
    shuffling
  • Key ExoIII digestion
  • Limited to two genes, one C/O per gene

Ostermeier, et al., Nat. Biotech. 1999, 17,
1205-1209.
6
SCRATCHY
  • SCRATCHY ITCHY DNA Shuffling
  • Multiple C/Os between two genes

Lutz et al., PNAS, 2001, 98, 11248-11253.
7
SCRATCHY
  • Enhanced crossover SCRATCHY uses the
    amplification step to skew the library population
    toward sections that already have C/Os

Lutz et al., PNAS, 2001, 98, 11248-11253.
8
Summary of Recombination Methods
  • ITCHY
  • More diverse set of functional fusions than DNA
    shuffling
  • Key step time-dependent digest
  • Limited to two genes, one C/O per gene
  • SCRATCHY
  • Two-gene hybrid assembly followed by shuffling
  • Shuffle ITCHY library ? increased of C/O

9
Goals
  • Demonstrate that low-homology recombination can
    be utilized to develop novel protein functions
    and that it represents an attractive method for
    therapeutic purposes
  • Use a combination of homology-dependent and
    homology-independent methods for the construction
    of chimeric theta-class GSTs.
  • Examine chimeric libraries by flow cytometry
  • Investigate the kinetics and substrate
    specificity of the newly generated chimeras and
    compare them to the parent enzymes

10
GST Enzymes
  • Crucial role in cellular detoxification
  • Antioxidant, antitoxin, essential cofactor for
    antioxidant enzymes
  • Depletion leads to cell death and degenerative
    conditions
  • GSH glutathione (reduced form)
  • GSSG glutathione disulfide (oxidized form)
  • Classes
  • alpha, beta, delta, zeta, theta, mu, pi, sigma,
    tau, phi, omega
  • G-site (GSH-binding domain)
  • aa 79.2
  • ntd 74.5
  • H-site (electrophilic substrate binding domain)
  • aa 41.4
  • ntd 57.9
  • hGSTT1-1 (DCM) vs rGSTT2-2 (1-menaphthyl sulfate)
  • rGSTT2-2 has a higher activity with CMAC than
    hGSTT1-1

11
Chimeric GSTT Library Construction
  • hGSTT1 and rGSTT2 are parental donors for ITCHY
    library construction
  • RH-A5, RH-F2, HR-25, HR-216
  • ITCHY followed by SCRATCHY and shuffling ?
    Humanized library
  • SCR9 and SCR23

12
Selected Chimeric Genes
  • HR-25
  • aa 78-83 (ARDIRS) no homology to either parent
    gene (unknown origin)
  • SRC9
  • Three C/Os, features analogous to HR-216 and
    RH-F2
  • SRC23
  • Two C/Os, two point mutations (L113P and W234R)

13
Enzymatic Characterization - Kinetic Parameters
  • rGSTT2-2 possess a 400-fold higher kcat/Km for
    CMAC than hGSTT1-1
  • r-hGSTT library ? RH-A5 (30) and RH-F2 (50)
    lower than rat
  • h-rGSTT library ? HR-216 (15 lower) and HR-25
    (equal)
  • SCR23 (90) and SCR9 (100) were the two most
    fluorescent clones

14
Enzymatic Characterization Substrate Specificity
  • CMAC 7-amino-4-chloromethyl coumarin
  • CDNB 1-chloro-2,4-dinitrobenzene

15
Enzymatic Characterization Substrate Specificity
  • PEITC phenethyl isothiocyanate
  • The N-terminal domain is indirectly enhancing the
    rat-like catalytic efficiency - HR-216 increased
    with PEITC (300) and CDNB (280)
  • SCR9 (CDNB) and SCR23 (EA) exhibit unique
    selectivity profiles

16
Conclusions
  • Did the authors achieve their goals?
  • Examine chimeric libraries (constructed via ITCHY
    and SCRATCHY) by flow cytometry
  • Investigate the kinetics and substrate
    specificity of the newly generated chimeras and
    compare them to the parent enzymes
  • Demonstrate that low-homology recombination can
    be utilized to develop novel protein functions
    and that it represents an attractive method for
    therapeutic purposes

?
?
?
17
Ongoing and Future Research
  • Continue to use homology-independent and
    low-homology recombination methods to develop
    proteins with novel functions.
  • Reduce the immunogenicity of enzymes for
    therapeutic purposes
  • Structural determination of SCR9 and SCR23
  • Substrate specificity of theta-class GSTs

18
Acknowledgments
  • Professor Huimin Zhao
  • Alexis Black and Teresa Fraterman
  • Professor Chad Rienstra
  • Kathy Lankster
  • All of you for listening !!!

19
Discussion Question
  • the current results indicate that the rat
    enzymes promiscuous nature is not due to a
    loose active site that indiscriminately
    accommodates aromatic compounds with reactive
    benzyl halides rather, it derives from the
    presence of distinct determinates for various
    halogenated target molecules.
  • Question To what distinct determinates are
    the authors referring to and how might they apply
    this new information toward future research?
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