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Updated: Sep 21, 2025
08:59
Defining Substrate Specificities for Lipase and Phospholipase Candidates
Published on: November 23, 2016
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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.
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.
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.
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