Comparative analysis of the metal-dependent structural and functional properties of mouse and human SMP30

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.衰老标记蛋白（Senescence Marker Protein, SMP30）是一类金属酶，在小鼠等非灵长类哺乳动物的抗坏血酸（ascorbic acid, AA）生物合成通路中展现出内酯酶活性。然而，包括人类在内的灵长类动物无法进行抗坏血酸的生物合成。已有多项研究证实，SMP30在哺乳动物体内参与维持钙稳态。此外，已有报道显示其可作用于有机磷酸酯（organophosphate, OP）底物，表现出混杂酶活性。本研究旨在通过重组表达与纯化小鼠及人类来源的SMP30蛋白，探究不同二价金属离子存在时二者的结构变化与功能偏差。为此，我们将小鼠SMP30（MoSMP30）与人类SMP30（HuSMP30）克隆至细菌表达载体中。鉴于上述蛋白主要以不溶性组分形式表达，我们分别从可溶性组分与包涵体中完成了蛋白的过表达与纯化。借助圆二色（circular dichroism, CD）光谱技术，我们分析了不同二价阳离子（Ca²+、Co²+、Mg²+及Zn²+）存在下纯化蛋白的折叠构象。结果显示，MoSMP30与HuSMP30均呈现天然折叠构象。为研究金属结合亲和力，我们通过紫外-可见（UV-VIS）滴定曲线计算了解离常数（Kd值），结果表明MoSMP30与Zn²+的结合亲和力最高；而HuSMP30则对Ca²+表现出最高结合亲和力，这提示HuSMP30在维持钙稳态中具有重要意义。我们以γ-硫代丁内酯和内吸磷-S（Demeton-S）为底物，在不同二价阳离子存在下开展了酶动力学实验。有趣的是，两种蛋白在Ca²+存在时均展现出内酯酶活性；此外，MoSMP30与HuSMP30分别在Co²+和Zn²+存在时表现出内酯酶活性。进一步研究发现，两种蛋白在Ca²+及Zn²+存在时均具有有机磷酸酯水解酶活性，这表明SMP30的混杂酶活性依赖于金属离子种类。