Evolution of Highly Active Enzymes by Homology-Independent Recombination

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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
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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

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Techniques

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

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The ITCHY and SCRATCHY Show !!!
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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.
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SCRATCHY

• SCRATCHY ITCHY DNA Shuffling
• Multiple C/Os between two genes

Lutz et al., PNAS, 2001, 98, 11248-11253.
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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.
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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

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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

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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

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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

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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)

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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

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Enzymatic Characterization Substrate Specificity

• CMAC 7-amino-4-chloromethyl coumarin
• CDNB 1-chloro-2,4-dinitrobenzene

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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

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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

?
?
?
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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

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Acknowledgments

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

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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?