Abstract：
The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin-dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions-but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein\'s C-terminal 23 residues (the \'α-peptide\'). Here, we relate the redox-induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (-SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys-SH difference signal to Cys88 and Cys494, both being remote from the moving α-peptide and the redox-active flavin cofactor. Yet, when the α-peptide is proteolytically removed, the Cys-SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α-peptide \'transforms\' the catalytically complex EcPOX into the catalytically \'simpler\' LpPOX.
摘要：
来自大肠杆菌(EcPOX)和植物乳杆菌(LpPOX)的丙酮酸氧化酶都是硫胺素依赖性黄素酶。它们的序列和结构密切相关，它们催化类似的反应——但它们的活性模式不同：LpPOX总是高活性的，EcPOX仅在被脂质或有限的蛋白水解激活时，两者都涉及蛋白质的C端23个残基(“α肽”)。这里,我们将EcPOX的氧化还原诱导的红外（IR）差异光谱与其异常活化机理联系起来。EcPOX的IR差异光谱以蛋白质骨架的贡献为标志，反映主要构象变化。罕见的巯基(-SH)差异信号表明半胱氨酸附近的变化。我们可以将Cys-SH差异信号固定到Cys88和Cys494，它们都远离移动的α肽和氧化还原活性黄素辅因子。然而，当α-肽被蛋白水解去除时，Cys-SH差异信号消失，以及酰胺范围内的几个差异信号。永久激活的EcPOXΔ23的其余IR特征与LpPOX的更简单特征非常相似。α-肽的丢失将催化复合物EcPOX“转化”为催化“更简单”的LpPOX。
