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In vitro and in silico characterization of a novel glutamate carboxypeptidase from Cohnella sp. A01
Seyed Mahdi Naeemi1, Saeed Aminzadeh2, Soyar Sari1
1Department of Molecular and Cellular Sciences, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Abstract:
Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 μM and 687 μmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 °C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 °C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.
Insights
A novel bacterial enzyme, recombinant glutamate carboxypeptidase (rCP302), efficiently degrades folic acid. Its stability and characteristics suggest potential pharmaceutical applications.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- Glutamate carboxypeptidase is a bacterial metallopeptidase.
- It hydrolyzes glutamate residues from the C-terminus of folic acid.
Purpose of the Study:
- To evaluate the heterologous production, purification, and characterization of a novel zinc-dependent glutamate carboxypeptidase (rCP302) from Cohnella sp. A01.
- To analyze its activity, structure, and stability under various conditions.
- To compare rCP302 with related enzymes.
Main Methods:
- Bioinformatic analysis for enzyme family classification.
- Heterologous production and purification.
- Enzyme activity assays at varying temperatures and pH.
- Fluorescence spectroscopy for structural elucidation.
- Kinetic parameter determination (Km, specific activity).
- Enzyme inhibition studies using EDTA.
- Half-life determination at different temperatures.
Main Results:
- rCP302 showed maximum similarity to the M20 family of metallopeptidases.
- Purified enzyme molecular weight was approximately 41.6 kDa.
- Optimal activity observed at 50°C and pH 7.2.
- Km and specific activity for folate were 0.108 μM and 687 μmol/min/mg, respectively.
- Enzyme activity was inhibited by zinc ion chelation with EDTA.
- rCP302 exhibited a long half-life (2012 min at 30°C).
Conclusions:
- rCP302 is a stable, zinc-dependent glutamate carboxypeptidase with high catalytic efficiency.
- Its favorable characteristics, including stability at mammalian physiological temperatures, warrant further investigation for pharmaceutical applications, particularly in folic acid metabolism modulation.
