Flavin redox state triggers conformational changes in the PutA protein from Escherichia coli.
Flavin redox state triggers conformational changes in the PutA protein from Escherichia coli.
复制标题
黄素氧化还原状态触发大肠杆菌 PutA 蛋白的构象变化。
DOI：
10.1021/bi0272196
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Becker,DonaldF
中科院分区：
文献类型：
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作者：
Zhu,Weidong;Becker,DonaldF
大肠杆菌中脯氨酸利用的调节涉及 PutA 黄素蛋白从细胞质到膜的外围位置的脯氨酸依赖性易位。在细胞质中，PutA 抑制脯氨酸利用 (put) 基因的转录，而膜结合的 PutA 则催化 1-脯氨酸氧化为谷氨酸。 PutA 从 DNA 结合蛋白转变为膜结合酶的机制涉及脯氨酸诱导的构象变化，其特征是在脯氨酸还原的 PutA 的有限蛋白水解过程中出现 119 kDa 片段。为了确定 FAD 氧化还原态是否是脯氨酸诱导的 PutA 构象变化的原因，我们通过有限的胰凝乳蛋白酶水解来区分 FAD 还原和脯氨酸类似物结合对 PutA 构象的影响。 PutA 的受控电位蛋白水解表明，119 kDa 带的形成发生在 -0.058 V (pH 7.5) 的 Em(conf) 值下，该值与与 PutA 结合的 FAD 的 Em 值相差 20 mV 以内。通过用 FAD 类似物 5-deazaFAD 重构 PutA 来操纵 Em(conf) 值，证实在脯氨酸存在下观察到的构象变化仅取决于 FAD 氧化还原状态。脯氨酸类似物，l-四氢-2-糠酸 (l-THFA)，在胰凝乳蛋白酶裂解 PutA 期间未能引发 119-kDa 片段的形成。相反，观察到约 93 kDa 的独特片段，表明独特的 PutA 构象异构体被 1-THFA 稳定。然而，1-THFA 复合的 PutA 的还原再生了 119-kDa 片段，表明 FAD 辅因子的还原覆盖了 1-THFA 诱导的构象变化。 PutA 中蛋白酶敏感性位点的绘图表明，由 FAD 还原和 I-THFA 结合引起的构象变化被传递到脯氨酸脱氢酶活性位点之外的结构域。
The regulation of proline utilization inEscherichia coliinvolves the proline-dependent translocation of the PutA flavoprotein from the cytoplasm to a peripheral position on the membrane. In the cytoplasm, PutA represses transcription of the proline utilization (put) genes while membrane-bound PutA catalyzes the oxidation ofl-proline to glutamate. The mechanism by which PutA switches from a DNA-binding protein to a membrane-bound enzyme involves a proline-induced conformational change that is characterized by the appearance of a 119-kDa fragment during limited proteolysis of proline-reduced PutA. To establish whether the FAD redox state is responsible for the proline-induced conformational change in PutA, we distinguished the effects that FAD reduction and proline analogue binding have on PutA conformation by limited chymotrypsin proteolysis. Controlled potentiometric proteolysis of PutA demonstrated that the formation of the 119-kDa band occurs at anEm(conf) value of −0.058 V (pH 7.5), which is within 20 mV of theEmvalue for FAD bound to PutA. The manipulation of theEm(conf) value by reconstitution of PutA with the FAD analogue, 5-deazaFAD, confirmed that the conformational change observed in the presence of proline is solely dependent on the FAD redox state. The proline analogue,l-tetrahydro-2-furoic acid (l-THFA), failed to elicit the formation of the 119-kDa fragment during chymotrypsin cleavage of PutA. Instead, a unique fragment of about 93-kDa was observed, indicating that a distinct PutA conformer is stabilized byl-THFA. Reduction ofl-THFA-complexed PutA, however, regenerated the 119-kDa fragment showing that reduction of the FAD cofactor overrides conformational changes induced byl-THFA. Mapping of the protease susceptibility sites in PutA revealed that the conformational changes caused by FAD reduction andl-THFA binding are transmitted to domains outside the proline dehydrogenase active site.