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Updated: May 26, 2026
Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)
Published on: December 20, 2010
Identification of Rv0535 as methylthioadenosine phosphorylase from Mycobacterium tuberculosis
Kajal Buckoreelall1, Yanjie Sun, Judith V Hobrath
1Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294, USA.
Abstract:
5'-methylthioadenosine (MTA) is a natural purine that is metabolized by methylthioadenosine phosphorylase (MTAP, E.C 2.4.2.28) in Eukarya and Archaea but generally not in bacteria. In this work, Rv0535, which has been annotated as a probable MTAP in Mycobacterium tuberculosis, was expressed in and purified from Escherichia coli BL21 (DE3). The purified protein displayed properties of a phosphorylase and MTA was the preferred substrate. Adenosine and S-adenosyl-l-homocysteine were poor substrates and no activity was detected with 5'-methylthioinosine, the other natural purines, or the natural pyrimidines. Kinetic analysis of M. tuberculosis MTAP showed that the K(m) value for MTA was 9 microM. Rv0535 was estimated as a 30 kDa protein on a denaturing SDS-PAGE gel, which agreed with the molecular mass predicted by its gene sequence. Using gel filtration chromatography, the native molecular mass of the enzyme was determined to be 60 ± 4 kDa, and thus indicated that M. tuberculosis MTAP is a dimer. Differences in active site between mycobacterial and human MTAPs were identified by homology modeling based on the crystal of the human enzyme. A complete structure-activity relationship analysis could identify differences in substrate specificity between the two enzymes to aid in the development of purine-based, anti-tuberculosis drugs.
Insights
Methylthioadenosine phosphorylase (MTAP) from Mycobacterium tuberculosis was purified and characterized. This enzyme specifically metabolizes 5'-methylthioadenosine (MTA), offering potential for developing novel anti-tuberculosis drugs.
Area of Science:
- Biochemistry
- Enzymology
- Structural Biology
Background:
- 5'-methylthioadenosine (MTA) is a natural purine metabolized by methylthioadenosine phosphorylase (MTAP) in eukaryotes and archaea, but not typically in bacteria.
- MTAP plays a role in purine metabolism, and its inhibition is a potential target for therapeutic intervention.
Purpose of the Study:
- To express, purify, and characterize the putative MTAP enzyme (Rv0535) from Mycobacterium tuberculosis.
- To investigate the substrate specificity and kinetic properties of M. tuberculosis MTAP.
- To explore structural differences between M. tuberculosis MTAP and human MTAP for drug development.
Main Methods:
- Recombinant expression and purification of Rv0535 from E. coli.
- Enzyme activity assays to determine substrate specificity and kinetic parameters (Km).
- SDS-PAGE and gel filtration chromatography for molecular mass determination.
- Homology modeling based on human MTAP crystal structure.
Main Results:
- Purified Rv0535 exhibited phosphorylase activity with MTA as the preferred substrate.
- Kinetic analysis revealed a Km value of 9 µM for MTA.
- M. tuberculosis MTAP was determined to be a dimer (native mass ~60 kDa).
- Homology modeling identified potential differences in the active site compared to human MTAP.
Conclusions:
- M. tuberculosis Rv0535 is a functional MTAP with specific substrate preferences.
- Understanding the structural and functional differences of M. tuberculosis MTAP can guide the design of targeted anti-tuberculosis agents.
- This research provides a basis for developing purine-based drugs against tuberculosis.
