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Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Christos S Karamitros1, Kyle Murray2, Brent Winemiller1

  • 1Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712.

Proceedings of the National Academy of Sciences of the United States of America
|June 3, 2022
PubMed
Summary

Directed evolution of Homo sapiens kynureninase (HsKYNase) by mutating flexible regions enhanced catalytic activity 45-fold. Distal mutations allosterically modulated enzyme flexibility, improving the rate-limiting chemical step.

Keywords:
HDX-MSMD simulationscatalysisenzyme engineeringintrinsic flexibility

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Area of Science:

  • Enzymology
  • Protein Engineering
  • Biochemistry

Background:

  • Enzyme dynamics across various timescales are crucial for substrate binding, catalysis, and product release.
  • The potential of exploiting conformational flexibility for directed enzyme evolution remains largely unexplored.

Purpose of the Study:

  • To investigate if targeting flexible regions distal to the active site can enhance enzyme catalytic activity.
  • To understand the mechanistic basis for improved catalysis in evolved enzymes.

Main Methods:

  • Site-directed mutagenesis of flexible regions in Homo sapiens kynureninase (HsKYNase).
  • Pre-steady-state kinetic analysis of wild-type and variant enzymes.
  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations.

Main Results:

  • A variant (BF-HsKYNase) with a 45-fold increased rate for the chemical step toward kynurenine was generated.
  • Distal mutations (>10 Å from the active site) significantly impacted the rate-limiting step.
  • Mutations allosterically altered the flexibility of the pyridoxal-5′-phosphate (PLP) binding pocket.

Conclusions:

  • Mutagenesis of distal flexible regions is a viable strategy for enhancing enzyme catalytic activity.
  • Allosteric modulation of enzyme flexibility, particularly in cofactor binding sites, can accelerate the catalyzed reaction.
  • Altering the conformational ensemble through distal mutations can lead to improved enzyme function.