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Published on: April 26, 2019
Heterologous expression and biochemical characterization of recombinant alpha phosphoglucomutase from Mycobacterium
Gagan Chhabra1, Divya Mathur, Aparna Dixit
1Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi, India.
Abstract:
Phosphoglucomutase (PGM) plays an important role in polysaccharide capsule formation and virulence in a number of bacterial pathogens. However, the enzyme has not yet been characterized from Mycobacterium tuberculosis (Mtb). Here, we report the biochemical properties of recombinant Mtb-PGM as well as the in silico structural analysis from Mtb H37Rv. The purified recombinant enzyme was enzymatically active with a specific activity of 67.5 U/mg and experimental k(cat) of 70.31 s(-1) for the substrate glucose-1-phosphate. The enzyme was stable in pH range 6.5-7.4 and exhibited temperature optima range between 30 and 40°C. Various kinetic parameters and constants of the rPGM were determined. A structural comparison of Modeller generated 3D Mtb-PGM structure with rabbit muscle PGM revealed that the two enzymes share the same overall heart shape and four-domain architecture, despite having only 17% sequence identity. However, certain interesting differences between the two have been identified, which provide an opportunity for designing new drugs to specifically target the Mtb-PGM. Also, in the absence of the crystal structure of the Mtb-PGM, the modeled structure could be further explored for in silico docking studies with suitable inhibitors.
Insights
Researchers characterized phosphoglucomutase (PGM) from Mycobacterium tuberculosis (Mtb). This study details the enzyme's biochemical properties and structural analysis, offering potential for new anti-TB drug development.
Area of Science:
- Biochemistry
- Structural Biology
- Microbiology
Background:
- Phosphoglucomutase (PGM) is crucial for polysaccharide capsule formation and virulence in bacterial pathogens.
- PGM from Mycobacterium tuberculosis (Mtb) has not been previously characterized.
- Understanding Mtb-PGM is vital for developing targeted anti-tuberculosis therapies.
Purpose of the Study:
- To biochemically characterize recombinant Mtb-PGM.
- To perform in silico structural analysis of Mtb-PGM.
- To identify potential drug targets within Mtb-PGM.
Main Methods:
- Expression and purification of recombinant Mtb-PGM.
- Enzymatic activity assays and determination of kinetic parameters (kcat, Km).
- In silico structural modeling using Modeller and comparison with known PGM structures.
Main Results:
- Recombinant Mtb-PGM exhibited specific activity of 67.5 U/mg and kcat of 70.31 s(-1).
- The enzyme demonstrated stability in a pH range of 6.5-7.4 and optimal temperature between 30-40°C.
- Structural analysis revealed conserved architecture despite low sequence identity (17%) with rabbit muscle PGM, highlighting unique differences.
Conclusions:
- Mtb-PGM is enzymatically active and possesses specific biochemical properties.
- The modeled 3D structure of Mtb-PGM provides a basis for further in silico studies.
- Identified structural differences present opportunities for designing novel Mtb-specific inhibitors, potentially leading to new anti-TB drugs.
