Overexpression, isotopic labeling, and spectral characterization of Enterobacter cloacae nitroreductase

Ronald L. Koder, Anne Frances Miller

Research output: Contribution to journalArticlepeer-review

18 Scopus citations


Bacterial nitroreductases have generated much interest recently due to their central roles in both nitroaromatic bioremediation and nitroaromatic toxicity, mutagenicity, and carcinogenicity. Enterobacter cloacae nitroreductase (NR) has been subcloned into the pET overexpression system and purified to homogeneity via a four-step procedure resulting in a final yield of 65.7 mg per liter. Overexpression in minimal media containing 15NH4C1 as the sole source of nitrogen yielded 37.6 mg per liter of homogenous NR containing >99 atom % 15N. A series of melting curves generated under a variety of solvent conditions established the optimal conditions for NR stability as pH 7.5, low ionic strength phosphate buffer. A two-dimensional 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance spectrum demonstrates this enzyme to be amenable to study by high-resolution multidimensional NMR in combination with amino-acid-specific isotopic labeling. Optical spectra of the purified enzyme suggest that the noncovalently bound flavin mononucleotide cofactor binds in a hydrophobic environment and is in the neutral and anionic protonation states in the oxidized and two-electron reduced oxidation states, respectively. NR exhibits a novel visible region circular dichroism spectrum which has a small distinct negative band at 366 nm and a large positive ellipticity at 454 nm with a shoulder centered at 480 nm.

Original languageEnglish
Pages (from-to)53-60
Number of pages8
JournalProtein Expression and Purification
Issue number1
StatePublished - Jun 1998

Bibliographical note

Funding Information:
This work was supported by PRF Grant ACS-PRF 28379 (to A.F.M.) and a National Science Foundation Graduate Research Fellowship (to R.L.K.). 1To whom correspondence should be addressed.

Funding Information:
Circular dichroism experiments were performed in the Biocalori-metry Center, a Biomedical Research Technology Resource Center sponsored by the National Institutes of Health (Grant RR-04328). The authors thank Dr. Maurice Bessman and Dr. David Frick of the Department of Biology, Johns Hopkins University, for the use of cloning facilities and instruction in their use and Dr. Chris Bryant of Centeon, L.L.C., for the gift of pCB18 and many helpful discussions.


  • Flavoprotein
  • Isotopic labeling
  • Nitroreductase

ASJC Scopus subject areas

  • Biotechnology


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