Ollagnier-de Choudens Sandrine, Sanakis Yiannis, Hewitson Kirsty S, Roach Peter, Münck Eckard, Fontecave Marc
Laboratoire de Chimie et Biochimie des Centres Rédox Biologiques, Departement de Biologie Moléculaire et Structurale-Chimie Biologie, CEA/CNRS/Université Joseph Fourier, UMR 5047, Grenoble, France.
J Biol Chem. 2002 Apr 19;277(16):13449-54. doi: 10.1074/jbc.M111324200. Epub 2002 Feb 7.
Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a 4Fe-4S cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and Mössbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 4Fe-4S cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 4Fe-4S cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the 4Fe-4S cluster.
生物素合酶（BioB）催化在脱硫生物素的C6和C9碳原子之间插入一个硫原子。来自大肠杆菌的重组BioB含有一个4Fe-4S簇，该簇被认为参与了S-腺苷甲硫氨酸（AdoMet）的还原和裂解，生成甲硫氨酸和负责从脱硫生物素上夺取氢原子的活性5'-脱氧腺苷自由基。通过电子顺磁共振（EPR）、穆斯堡尔光谱以及甲硫氨酸定量分析，我们证明了还原态的S = 1/2 4Fe-4S簇确实能够向AdoMet注入一个电子，生成等量的甲硫氨酸和S = 0 4Fe-4S簇。该反应不需要脱硫生物素。通过定点诱变我们还表明，在BioB的八个半胱氨酸中，只有三个（Cys-53、Cys-57、Cys-60）对于AdoMet的还原裂解是必需的，这表明这些半胱氨酸参与了4Fe-4S簇的螯合。
