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

doi:10.1016/j.pep.2004.01.005

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 journal › Article › peer-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 language	English (US)
Pages (from-to)	131-141
Number of pages	11
Journal	Protein Expression and Purification
Volume	35
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2004.01.005
State	Published - May 2004
Externally published	Yes

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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10.1016/j.pep.2004.01.005

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

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.",

keywords = "Bacillus subtilis, Cupin, Dioxygenase, Electron paramagnetic resonance, Iron, Non-heme iron enzymes, Quercetin",

author = "Barney, {Brett M.} and Schaab, {Matthew R.} and Russell LoBrutto and Francisco, {Wilson A.}",

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.",

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doi = "10.1016/j.pep.2004.01.005",

language = "English (US)",

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T2 - Purification and characterization of an iron-containing quercetin 2,3-dioxygenase from Bacillus subtilis

AU - Barney, Brett M.

AU - Schaab, Matthew R.

AU - LoBrutto, Russell

AU - Francisco, Wilson A.

N1 - 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.

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N2 - 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.

AB - 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.

KW - Bacillus subtilis

KW - Cupin

KW - Dioxygenase

KW - Electron paramagnetic resonance

KW - Iron

KW - Non-heme iron enzymes

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ER -

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

Abstract

Bibliographical note

Keywords

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