Evidence for a new metal in a known active site: purification and characterization of an iron-containing quercetin 2,3-dioxygenase from Bacillus subtilis.
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.
Barney
Brett M
BM
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Schaab
Matthew R
MR
LoBrutto
Russell
R
Francisco
Wilson A
WA
D001230:Aspergillus / Q000201:enzymology; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D001665:Binding Sites; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D004791:Enzyme Inhibitors / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D015394:Molecular Structure; D010105:Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011794:Quercetin / Q000378:metabolism
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D021122:Protein Subunits; D011794:Quercetin; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C003281:quercetin 2,3-dioxygenase