Evidence for a new metal in a known active site: Purification and characterization of an iron-containing quercetin 2,3-dioxygenase from Bacillus subtilis

Brett M. Barney, Matthew R. Schaab, Russell LoBrutto, Wilson A. Francisco

Research output: Contribution to journalArticlepeer-review

45 Scopus citations

Abstract

The protein YxaG from Bacillus subtilis, of previously unknown function, was found to have quercetin 2,3-dioxygenase activity when overexpressed in Escherichia coli. The enzyme converts the flavonol quercetin to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide, indicating that it performs the same reaction and yields the same products as the well-characterized copper-containing quercetin 2,3-dioxygenase from Aspergillus. In contrast to the Aspergillus protein, YxaG contains iron, and the enzyme is sensitive to strong Fe(II) chelators, similar to the extensively studied catechol dioxygenases. The active site metal was probed by EPR spectroscopy using the label nitric oxide to confirm the presence of an Fe(II) atom. The kinetic parameters and pH activity profiles are also markedly different from those of the copper-containing quercetin 2,3-dioxygenases from Aspergillus. YxaG represents the first example of a prokaryotic quercetin 2,3-dioxygenase.

Original languageEnglish (US)
Pages (from-to)131-141
Number of pages11
JournalProtein Expression and Purification
Volume35
Issue number1
DOIs
StatePublished - May 2004
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported in part by NSF-Grant MCB-0317126 (to W.A.F.). B.M.B. was supported in part by a fellowship through the Research Training Group in Optical Biomolecular Devices provided under a grant from the NSF (DB1-9602258-003). The Voyager DE STR mass spectrometer used by the Protein Laboratory Facility at Arizona State University was purchased under NSF Grant CHE-0131222. We also wish to thank John C. Lopez and Daniel C. Brune for analysis of YxaG by MALDI-TOF, Thomas Colella for assistance with atomic absorption analysis, William Burke for providing the strain and information about Bacillus subtilis , and Scott Bingham for sequencing the cloned gene in the plasmid pQUER4.

Keywords

  • Bacillus subtilis
  • Cupin
  • Dioxygenase
  • Electron paramagnetic resonance
  • Iron
  • Non-heme iron enzymes
  • Quercetin

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