Kinetic and Spectroscopic Studies on the Quercetin 2,3-Dioxygenase from Bacillus subtilis

详细信息    查看全文

• 作者：Matthew R. Schaab ; Brett M. Barney ; and Wilson A. Francisco
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：January 24, 2006
• 年：2006
• 卷：45
• 期：3
• 页码：1009 - 1016
• 全文大小：97K
• 年卷期：v.45,no.3(January 24, 2006)
• ISSN：1520-4995

文摘

Quercetin 2,3-dioxygenase from Bacillus subtilis (QueD) converts the flavonol quercetin andmolecular oxygen to 2-protocatechuoylphloroglucinolcarboxylic acid and carbon monoxide. QueD, theonly known quercetin 2,3-dioxygenase from a prokaryotic organism, has been described as an Fe²⁺-dependent bicupin dioxygenase. Metal-substituted QueDs were generated by expressing the enzyme inEscherichia coli grown on minimal media in the presence of a number of divalent metals. The additionof Mn²⁺, Co²⁺, and Cu²⁺ generated active enzymes, but the addition of Zn²⁺, Fe²⁺, and Cd²⁺ did notincrease quercetinase activity to any significant level over a control in which no divalent ions were addedto the media. The Mn²⁺- and Co²⁺-containing QueDs were purified, characterized by metal analysis andEPR spectroscopy, and studied by steady-state kinetics. Mn²⁺ was found to be incorporated nearlystoichiometrically to the two cupin motifs. The hyperfine coupling constant of the g = 2 signal in theEPR spectra of the Mn²⁺-containing enzyme showed that the two Mn²⁺ ions are ligated in an octahedralcoordination. The turnover number of this enzyme was found to be in the order of 25 s^-1, nearly 40-foldhigher than that of the Fe²⁺-containing enzyme and similar in magnitude to that of the Cu²⁺-containingquercertin 2,3-dioxygenase from Aspergillus japonicus. In addition, kinetic and spectroscopic data suggestthat the catalytic mechanism of QueD is different from that of the Aspergillus quercetinases but similarto that proposed for the extradiol catechol dioxygenases. This study provides evidence that Mn²⁺ mightbe the preferred cofactor for this enzyme and identifies QueD as a new member of the manganesedioxygenase family.