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Identifying and Engineering Flavin Dependent Halogenases for Selective Biocatalysis.

Jared C Lewis1

  • 1Department of Chemistry, Indiana University, Bloomington, Indiana 47405, United States.

Accounts of Chemical Research
|July 22, 2024
PubMed
Summary
This summary is machine-generated.

Engineered flavin-dependent halogenases (FDHs) enable selective halogenation of diverse non-native compounds. Directed evolution and genome mining expanded FDH utility for biocatalytic synthesis, including enantioselective reactions.

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Area of Science:

  • Biocatalysis and Enzyme Engineering
  • Organic Chemistry
  • Synthetic Biology

Background:

  • Organohalogen compounds are vital in pharmaceuticals and agrochemicals, but their synthesis often requires harsh conditions and multistep processes.
  • Flavin-dependent halogenases (FDHs) offer a nature-inspired solution for selective halogenation, utilizing FADH2 and molecular oxygen.
  • Previous FDH engineering efforts via site-directed mutagenesis yielded limited improvements in substrate scope and selectivity for non-native substrates.

Purpose of the Study:

  • To overcome limitations in FDH engineering for preparative halogenation of non-native substrates.
  • To expand the utility of FDHs for diverse biocatalytic halogenation reactions beyond simple aromatic halogenation.
  • To develop predictive models for FDH selectivity and identify novel FDHs with unique catalytic capabilities.

Main Methods:

  • Optimization of expression conditions for FDH RebH and its reductase RebF.
  • Directed evolution of RebH to improve stability, substrate scope, and site selectivity.
  • X-ray crystallography, molecular dynamics simulations, and family-wide genome mining to identify and characterize diverse FDHs.
  • Exploration of engineered FDHs in enantioselective desymmetrization, atroposelective halogenation, halocyclization, and reactions catalyzed by the single-component FDH/FRed AetF.

Main Results:

  • Engineered RebH demonstrated preparative halogenation of non-native substrates with catalyst-controlled selectivity.
  • Directed evolution significantly enhanced FDH stability, substrate scope, and site selectivity, achieving synthetically useful levels.
  • Genome mining identified diverse FDHs with novel substrate scopes and complementary regioselectivity for complex compounds.
  • FDHs were shown to catalyze enantioselective reactions, including desymmetrization, atroposelective halogenation, and halocyclization.
  • The single-component FDH/FRed AetF exhibited unique substrate halogenation capabilities and high enantioselectivity, including site-selective aromatic iodination and enantioselective iodoetherification.

Conclusions:

  • Engineered FDHs, developed through directed evolution and genome mining, represent powerful tools for biocatalytic halogenation.
  • These enzymes offer enhanced stability, broader substrate scope, and improved selectivity compared to wild-type counterparts.
  • The demonstrated utility in complex synthetic applications, including enantioselective transformations, highlights the significant promise of FDHs for future chemical synthesis.