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Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode for Catalytically Competent Nitroreductase.

Warintra Pitsawong1, Chad A Haynes2, Ronald L Koder1

  • 1Department of Chemistry, University of Kentucky, 505 Rose Street, Lexington, KY 40506-0055, USA.

Structure (London, England : 1993)
|June 6, 2017
PubMed
Summary

Nitroreductase enzymes efficiently reduce nitroaromatics for bioremediation and drug activation. Structural and kinetic studies reveal key hydride transfer steps and substrate orientation, refining our understanding of this versatile enzyme.

Keywords:
flavoenzymeisotope effectsnitroreductaseprodrug activationremediationstructuresubstrate binding mode

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Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Nitroreductase (NR) from Enterobacter cloacae is a versatile enzyme capable of reducing diverse nitroaromatic compounds.
  • NR has significant potential applications in bioremediation, prodrug activation, and enzyme-assisted synthesis.
  • Understanding the catalytic mechanism of NR is crucial for optimizing its applications.

Purpose of the Study:

  • To elucidate the detailed mechanism of nitroreductase (NR) from Enterobacter cloacae.
  • To determine the structural basis for NR's substrate specificity and catalytic activity.
  • To investigate the H-transfer steps involved in NR's two half-reactions.

Main Methods:

  • Crystal structures of NR complexes with bound substrate analogs were determined.
  • Kinetic isotope effect (KIE) measurements were employed to study H-transfer steps.
  • Mechanistic information was used to provide structural restraints for substrate orientation.

Main Results:

  • Crystal structures revealed NR complexes with bound substrate or analog for each half-reaction.
  • KIEs confirmed hydride transfer from NADH to the flavin and during p-nitrobenzoic acid reduction.
  • Structural data indicated a specific orientation of the nitro group relative to the flavin N5.
  • Solvent protonation was identified as a key step accommodating diverse nitro group placements.

Conclusions:

  • The study provides a revised mechanistic understanding of nitroreductase activity, emphasizing hydride transfer and solvent involvement.
  • Structural insights clarify substrate binding and orientation, explaining NR's broad substrate repertoire.
  • This work offers a foundation for protein engineering and application development of nitroreductases.