Faculty of Life Sciences and Biotechnology, South Asian University, New Delhi, India.
PLoS One. 2019 Jun 20;14(6):e0218629. doi: 10.1371/journal.pone.0218629. eCollection 2019.
Senescence Marker Protein (SMP30) is a metalloenzyme that shows lactonase activity in the ascorbic acid (AA) biosynthesis pathway in non-primate mammals such as a mouse. However, AA biosynthesis does not occur in the primates including humans. Several studies have shown the role of SMP30 in maintaining calcium homeostasis in mammals. In addition, it is also reported to have promiscuous enzyme activity with an organophosphate (OP) substrate. Hence, this study aims to recombinantly express and purify the SMP30 proteins from both mouse and human, and to study their structural alterations and functional deviations in the presence of different divalent metals. For this, mouse SMP30 (MoSMP30) as well as human SMP30 (HuSMP30) were cloned in the bacterial expression vector. Proteins were overexpressed and purified from soluble fractions as well as from inclusion bodies as these proteins were expressed largely in insoluble fractions. The purified proteins were used to study the folding conformations in the presence of different divalent cations (Ca2+, Co2+, Mg2+, and Zn2+) with the help of circular dichroism (CD) spectroscopy. It was observed that both MoSMP30 and HuSMP30 acquired native folding conformations. To study the metal-binding affinity, dissociation constant (Kd values) were calculated from UV-VIS titration curve, which showed the highest affinity of MoSMP30 with Zn2+. However, HuSMP30 showed the highest affinity with Ca2+, suggesting the importance of HuSMP30 in maintaining calcium homeostasis. Enzyme kinetics were performed with γ-Thiobutyrolactone and Demeton-S in the presence of different divalent cations. Interestingly, both the proteins showed lactonase activity in the presence of Ca2+. In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co2+ and Zn2+ respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca2+ as well as Zn2+, suggesting the metal-dependent promiscuous nature of SMP30.
衰老标志物蛋白 (SMP30) 是一种金属酶，在非灵长类哺乳动物（如小鼠）的抗坏血酸 (AA) 生物合成途径中表现出内酯酶活性。然而，AA 生物合成并不发生在包括人类在内的灵长类动物中。几项研究表明 SMP30 在哺乳动物维持钙稳态中发挥作用。此外，它还被报道具有与有机磷 (OP) 底物的混杂酶活性。因此，本研究旨在从鼠和人重组表达和纯化 SMP30 蛋白，并研究它们在不同二价金属存在下结构的改变和功能的偏差。为此，将鼠 SMP30 (MoSMP30) 和人 SMP30 (HuSMP30) 克隆到细菌表达载体中。蛋白质从可溶部分和包涵体中过量表达和纯化，因为这些蛋白质主要在不溶部分中表达。利用圆二色性 (CD) 光谱研究了不同二价阳离子（Ca2+、Co2+、Mg2+和 Zn2+）存在下蛋白质的折叠构象。结果表明，MoSMP30 和 HuSMP30 均获得了天然的折叠构象。为了研究金属结合亲和力，通过紫外-可见滴定曲线计算了解离常数 (Kd 值)，结果表明 MoSMP30 与 Zn2+ 具有最高的亲和力。然而，HuSMP30 与 Ca2+ 具有最高的亲和力，这表明 HuSMP30 在维持钙稳态中的重要性。在不同二价阳离子存在下，用γ-硫代丁内酯和 Demeton-S 进行酶动力学研究。有趣的是，两种蛋白质在 Ca2+ 存在下均表现出内酯酶活性。此外，MoSMP30 和 HuSMP30 分别在 Co2+ 和 Zn2+ 存在下也表现出内酯酶活性。此外，两种蛋白质在 Ca2+ 和 Zn2+ 存在下均表现出 OP 水解酶活性，这表明 SMP30 具有金属依赖性混杂酶活性。
