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

Autor: Parmanand Pandey, Nidhi Dhama, Roshan Kumar Dutta, Fauzia Parween, Md. Summon Hossain, Rinkoo D. Gupta
Rok vydání: 2019
Předmět:
0301 basic medicine
Circular dichroism
Disulfoton
Hydrolases
Protein Expression
Plasma protein binding
Biochemistry
Physical Chemistry
Mice
Database and Informatics Methods
Protein structure
Spectrum Analysis Techniques
4-Butyrolactone
Macromolecular Structure Analysis
chemistry.chemical_classification
Mice
Inbred BALB C
Multidisciplinary
Intracellular Signaling Peptides and Proteins
Amino acid
Enzymes
Chemistry
Circular Dichroism Spectroscopy
Physical Sciences
Medicine
Sequence Analysis
Protein Binding
Research Article
Protein Structure
Cations
Divalent
Bioinformatics
Science
Molecular Dynamics Simulation
Research and Analysis Methods
Divalent
03 medical and health sciences
Cations
Hydrolase
Gene Expression and Vector Techniques
Animals
Humans
Enzyme kinetics
Molecular Biology Techniques
Molecular Biology
Enzyme Kinetics
Ions
Molecular Biology Assays and Analysis Techniques
Binding Sites
030102 biochemistry & molecular biology
Sequence Homology
Amino Acid
Calcium-Binding Proteins
Biology and Life Sciences
Proteins
Ascorbic acid
Kinetics
030104 developmental biology
chemistry
Enzymology
Ultraviolet-Visible Spectroscopy
Sequence Alignment
Zdroj: PLoS ONE
PLoS ONE, Vol 14, Iss 6, p e0218629 (2019)
ISSN: 1932-6203
Popis: 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.
Databáze: OpenAIRE
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