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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 maintain...

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Detalles Bibliográficos
Autores principales: Dutta, Roshan Kumar, Parween, Fauzia, Hossain, Md. Summon, Dhama, Nidhi, Pandey, Parmanand, Gupta, Rinkoo Devi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586323/
https://www.ncbi.nlm.nih.gov/pubmed/31220150
http://dx.doi.org/10.1371/journal.pone.0218629
Descripción
Sumario: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 (Ca(2+), Co(2+), Mg(2+), and Zn(2+)) 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 Zn(2+). However, HuSMP30 showed the highest affinity with Ca(2+), 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 Ca(2+). In addition, MoSMP30 and HuSMP30 also showed lactonase activity in the presence of Co(2+) and Zn(2+) respectively. Moreover, both the proteins showed OP hydrolase activities in the presence of Ca(2+) as well as Zn(2+), suggesting the metal-dependent promiscuous nature of SMP30.