Barney Brett M, Schaab Matthew R, LoBrutto Russell, Francisco Wilson A
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604, USA.
Protein Expr Purif. 2004 May;35(1):131-41. doi: 10.1016/j.pep.2004.01.005.
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.
来自枯草芽孢杆菌的蛋白质YxaG，其功能此前未知，当在大肠杆菌中过表达时，被发现具有槲皮素2,3 -双加氧酶活性。该酶将黄酮醇槲皮素转化为2 -原儿茶酰间苯三酚羧酸和一氧化碳，这表明它与来自曲霉属的已充分表征的含铜槲皮素2,3 -双加氧酶执行相同的反应并产生相同的产物。与曲霉属蛋白质不同，YxaG含有铁，并且该酶对强亚铁螯合剂敏感，这与广泛研究的儿茶酚双加氧酶类似。使用一氧化氮标记通过电子顺磁共振光谱对活性位点金属进行探测，以确认亚铁原子的存在。其动力学参数和pH活性曲线也与来自曲霉属的含铜槲皮素2,3 -双加氧酶明显不同。YxaG代表了原核生物槲皮素2,3 -双加氧酶的首个实例。
