Serratia marcescens is an emerging health-threatening, gram-negative opportunistic pathogen associated with a wide variety of localized and life-threatening systemic infections. One of the most crucial virulence factors produced by S. marcescens is serratiopeptidase, a 50.2-kDa repeats-in-toxin (RTX) family broad-specificity zinc metalloprotease. RTX family proteins are functionally diverse exoproteins of gram-negative bacteria that exhibit calcium-dependent structural dynamicity and are secreted through a common type-1 secretion system (T1SS) machinery. To evaluate the impact of various divalent ligands on the folding and maturation of serratiopeptidase zymogen, the protein was purified and a series of structural and functional investigations were undertaken. The results indicate that calcium binding to the C-terminal RTX domain acts as a folding switch, triggering a disordered-to-ordered transition in the enzyme's conformation. Further, the auto-processing of the 16-amino acid N-terminal pro-peptide results in the maturation of the enzyme. The binding of calcium ions to serratiopeptidase causes a highly cooperative conformational transition in its structure, which is essential for the enzyme's activation and maturation. This conformational change is accompanied by an increase in solubility and enzymatic activity. For efficient secretion and to minimize intracellular toxicity, the enzyme needs to be in an unfolded extended form. The calcium-rich extracellular environment favors the folding and processing of zymogen into mature serratiopeptidase, i.e., the holo-form required by S. marcescens to establish infections and survive in different environmental niches.

中文翻译：

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钙诱导的结构转变对于沙肽酶酶原的折叠、功能和加工成成熟形式至关重要

粘质沙雷氏菌是一种新出现的威胁健康的革兰氏阴性机会致病菌，与多种局部和危及生命的全身感染有关。粘质链球菌产生的最重要的毒力因子之一是锯齿肽酶，它是一种 50.2 kDa 毒素重复序列 (RTX) 家族广泛特异性锌金属蛋白酶。RTX 家族蛋白是功能多样的革兰氏阴性菌外蛋白，表现出钙依赖性结构动态性，并通过常见的 1 型分泌系统 (T1SS) 机制分泌。为了评估各种二价配体对锯齿肽酶酶原折叠和成熟的影响，纯化了蛋白质并进行了一系列结构和功能研究。结果表明，钙与 C 端 RTX 结构域的结合充当折叠开关，引发酶构象从无序到有序的转变。此外，16 个氨基酸 N 端前肽的自动加工导致酶的成熟。钙离子与锯齿肽酶的结合导致其结构中高度协同的构象转变，这对于酶的激活和成熟至关重要。这种构象变化伴随着溶解度和酶活性的增加。为了有效分泌并最大限度地减少细胞内毒性，酶需要处于未折叠的延伸形式。富含钙的细胞外环境有利于酶原折叠和加工成成熟的锯齿肽酶，即粘质链球菌建立感染并在不同环境中生存所需的全型形式。