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Attempts to develop an enzyme converting DHIV to KIV.

Kenji Oki1,2, Frederick S Lee3, Stephen L Mayo1,4

  • 1Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd., MC 114-96, Pasadena, CA 91125, USA.

Protein Engineering, Design & Selection : PEDS
|December 25, 2019
PubMed
Summary
This summary is machine-generated.

Researchers explored replacing expensive dihydroxy-acid dehydratase (DHAD) with engineered sugar acid dehydratases for R-2,3-dihydroxyisovalerate (DHIV) dehydration. Computational protein design efforts did not yield enzymes with the desired DHIV dehydration activity.

Keywords:
computational protein designenolase superfamilyisobutanolmevalonate-3-kinasesugar acid dehydratase

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Computational Protein Design
  • Metabolic Engineering

Background:

  • Dihydroxy-acid dehydratase (DHAD) uses an oxidation-sensitive, costly Fe-S cluster to dehydrate R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV).
  • Sugar acid dehydratases offer a cost-efficient alternative, utilizing a magnesium ion for similar reactions.
  • The need for stable, economical biocatalysts for DHIV conversion is critical in various biotechnological applications.

Purpose of the Study:

  • To engineer a cost-efficient sugar acid dehydratase to replace the expensive DHAD for DHIV dehydration.
  • To investigate alternative substrate activation mechanisms, including chemical activation and phosphorylation, for enzyme engineering.
  • To explore the potential of computational protein design (CPD) in creating novel biocatalysts for specific metabolic pathways.

Main Methods:

  • Computational protein design (CPD) was employed to modify the binding pocket of sugar acid dehydratases.
  • Enzyme activity screening involved using chemically activated substrate analogs like chlorolactate (CLD) and assessing beta-elimination activity.
  • Investigated phosphorylation of DHIV using mevalonate-3-kinase (M3K) and attempted engineering of enzymes for the phosphorylated substrate.

Main Results:

  • Initial attempts to engineer sugar acid dehydratases for DHIV accommodation were unsuccessful.
  • Mandelate racemase (PpManR) and a putative sugar acid dehydratase (StPutD) showed activity towards CLD, with CPD improving PpManR's kcat/KM for CLD four-fold.
  • Engineered variants did not exhibit DHIV dehydration activity; PtM3K showed ATP hydrolysis with DHIV, suggesting phosphorylation, but engineered enzymes for 3-phospho-DHIV were not obtained.

Conclusions:

  • Direct engineering of sugar acid dehydratases for DHIV dehydration using CPD proved challenging.
  • While enzyme variants showed activity on analogs, they failed to catalyze the target DHIV dehydration reaction.
  • Exploration of alternative activation mechanisms like phosphorylation warrants further investigation for biocatalyst development.