Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway

Cory J. Knoot, Elena G. Kovaleva, John D. Lipscomb

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The diiron cluster-containing oxygenase CmlI catalyzes the conversion of the aromatic amine precursor of chloramphenicol to the nitroaromatic moiety of the active antibiotic. The X-ray crystal structures of the fully active, N-terminally truncated CmlIΔ33 in the chemically reduced Fe2+/Fe2+ state and a cisμ-1,2(η11)-peroxo complex are presented. These structures allow comparison with the homologous arylamine oxygenase AurF as well as other types of diiron cluster-containing oxygenases. The structural model of CmlIΔ33 crystallized at pH 6.8 lacks the oxo-bridge apparent from the enzyme optical spectrum in solution at higher pH. In its place, residue E236 forms a μ-1,3(η12) bridge between the irons in both models. This orientation of E236 stabilizes a helical region near the cluster which closes the active site to substrate binding in contrast to the open site found for AurF. A very similar closed structure was observed for the inactive dimanganese form of AurF. The observation of this same structure in different arylamine oxygenases may indicate that there are two structural states that are involved in regulation of the catalytic cycle. Both the structural studies and single crystal optical spectra indicate that the observed cis μ-1,2(η11)-peroxo complex differs from the μ-η12-peroxo proposed from spectroscopic studies of a reactive intermediate formed in solution by addition of O2 to diferrous CmlI. It is proposed that the structural changes required to open the active site also drive conversion of the µ-1,2-peroxo species to the reactive form.

Original languageEnglish (US)
Pages (from-to)589-603
Number of pages15
JournalJournal of Biological Inorganic Chemistry
Issue number5-6
StatePublished - Sep 1 2016

Bibliographical note

Funding Information:
This work was supported by the National Institutes of Health Grants GM 100943 and GM 118030 (to J.D.L.) and NIH graduate traineeship GM 08700 (to C. J. K.). We would like to thank Carrie Wilmot and her research group for many helpful discussions. We also thank Ed Hoeffner at the University of Minnesota Kahlert Structural Biology Laboratory for suggestions in indexing the CmlIΔ33 data and collecting the data sets. We also thank Klaus Lovendahl for generating the expression construct for the truncated enzyme and Anna Komor and Brent Rivard for assistance with biochemical characterization. Diffraction data were collected at Argonne National Laboratory, Structural Biology Center Beamline 19-ID at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the US Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. We are thankful for computational resources from the Supercomputing Institute and the facilities at the Kahlert Structural Biology Laboratory at the University of Minnesota.

Publisher Copyright:
© 2016, SBIC.


  • Antibiotic biosynthesis
  • Arylamine oxygenase
  • Diiron cluster
  • Non-heme iron
  • Oxygen activation
  • Peroxo intermediate


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