Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis
Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis
复制标题
DOI：
10.1074/jbc.m404381200
复制
发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
Booker, SJ
中科院分区：
文献类型：
--
作者：
Cicchillo, RM;Baker, MA;Booker, SJ
L-丝氨酸脱氨酶催化L-丝氨酸的脱氨，产生丙酮酸和氨。已经描述了这些蛋白质的两个家族，并通过各自在催化中使用的辅因子来描绘。这些是吡哆醛5 '-磷酸依赖性脱氨酶和在体外被铁和二硫苏糖醇激活的脱氨酶。与使用吡哆醛5 '-磷酸的酶相反，铁依赖性脱氨酶的详细物理和机械表征是有限的，主要是因为它们的极端不稳定性。我们在这里报告的L-丝氨酸脱氨酶的表征大肠杆菌，这是产品的sdaA基因。当厌氧纯化时，分离的蛋白质含有1.86 +/- 0.46当量的铁和0.670 +/- 0.019当量的硫化物/多肽，并显示出与[4Fe-4S]簇一致的UV-可见光谱。蛋白质与铁和硫化物的重构产生相当多的簇，并且用连二亚硫酸盐处理重构的蛋白质产生轴向EPR谱，显示g1 = 2.03和g（垂直于）= 1.93。Fe-57重组蛋白质的穆斯堡尔谱显示，大部分铁是以[4Fe-4S]（2+）簇的形式存在，如典型的穆斯堡尔参数-异构体位移δ = 0.47 mm/s，四极分裂Δ E（Q）= 1.14 mm/s和抗磁性（S = 0）基态所证明的。用L-丝氨酸处理连二亚硫酸盐还原的蛋白质导致在蛋白质的EPR谱中g = 2.03处的特征略微变宽，并且信号强度急剧下降，这表明氨基酸直接与簇相互作用。
L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5'-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5'-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 +/- 0.46 eq of iron and 0.670 +/- 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g\\ = 2.03 and g(perpendicular to) = 1.93. Mossbauer spectra of the Fe-57-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S](2+) clusters, as evidenced by the typical Mossbauer parameters-isomer shift, delta = 0.47 mm/s, quadrupole splitting of DeltaE(Q) = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.