Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.
Exploring the selenium-over-sulfur substrate specificity and kinetics of a bacterial selenocysteine lyase.
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DOI：
10.1016/j.biochi.2021.01.002
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发表时间：
2021-03
期刊：
影响因子：
3.9
通讯作者：
Self WT
中科院分区：
文献类型：
--
作者：
Johnstone MA;Nelson SJ;O'Leary C;Self WT
硒是许多生物体中重要的微量营养素。微量的硒是微生物利用的必需品，但过量的硒是有毒的，因此，硒可以被视为生物学上的“双刃剑”。硒在化学上与必需元素硫相似，但奇怪的是，进化选择了前者而不是后者作为氧化还原酶的子集。参与硫代谢的酶在防止硒掺入方面的歧视性较小;然而，其特异性掺入硒蛋白揭示了一个尚不完全了解的高度歧视性过程。我们已经确定了SclA，一种NifS样蛋白在医院病原体，粪肠球菌，其特征在于其酶活性和特异性的L-硒代半胱氨酸超过L-半胱氨酸。已知人硒代半胱氨酸裂解酶中硒代半胱氨酸特异性需要Asp-146。因此，使用计算生物学，我们比较了细菌和哺乳动物的酶，并确定了His-100，在SclA的Asp-146直系同源物，并产生定点突变体，以研究残基的潜在作用的L-硒代半胱氨酸歧视机制。过表达的蛋白质，纯化，并表征其生化特性。所有突变体对L-硒代半胱氨酸表现出不同的Michaelis-Menten行为，但没有发现His-100对这种活性是必需的。此外，L-半胱氨酸作为所有酶的竞争性抑制剂，具有比L-硒代半胱氨酸更高的亲和力。最后，我们发现SclA在L-半胱氨酸作为不良底物时表现出低活性，而与突变无关。我们的结论是，His-100是不需要L-硒代半胱氨酸特异性，强调细菌NifS样蛋白和哺乳动物硒代半胱氨酸裂解酶之间的歧视机制的固有差异。
Selenium is a vital micronutrient in many organisms. While traces are required for microbial utilization, excess amounts are toxic; thus, selenium can be regarded as a biological “double-edged sword”. Selenium is chemically similar to the essential element sulfur, but curiously, evolution has selected the former over the latter for a subset of oxidoreductases. Enzymes involved in sulfur metabolism are less discriminate in terms of preventing selenium incorporation; however, its specific incorporation into selenoproteins reveals a highly discriminate process that is not completely understood. We have identified SclA, a NifS-like protein in the nosocomial pathogen, Enterococcus faecalis, and characterized its enzymatic activity and specificity for L-selenocysteine over L-cysteine. It is known that Asp-146 is required for selenocysteine specificity in the human selenocysteine lyase. Thus, using computational biology, we compared the bacterial and mammalian enzymes and identified His-100, an Asp-146 ortholog in SclA, and generated site-directed mutants in order to study the residue’s potential role in the L-selenocysteine discrimination mechanism. The proteins were overexpressed, purified, and characterized for their biochemical properties. All mutants exhibited varying Michaelis-Menten behavior towards L-selenocysteine, but His-100 was not found to be essential for this activity. Additionally, L-cysteine acted as a competitive inhibitor of all enzymes with higher affinity than L-selenocysteine. Finally, we discovered that SclA exhibited low activity with L-cysteine as a poor substrate regardless of mutations. We conclude that His-100 is not required for L-selenocysteine specificity, underscoring the inherent differences in discriminatory mechanisms between bacterial NifS-like proteins and mammalian selenocysteine lyases.
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