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Updated: Jun 2, 2026
Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092
Published on: October 2, 2017
Structural and functional studies of mycobacterial IspD enzymes
Christofer Björkelid1, Terese Bergfors, Lena M Henriksson
1Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE-75124 Uppsala, Sweden.
Abstract:
A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.
Insights
Researchers targeted the IspD enzyme in the MEP pathway, crucial for pathogens like tuberculosis and malaria. Structural studies of Mycobacterium IspD provide tools for developing broad-spectrum inhibitors against these diseases.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Pathogens causing tuberculosis and malaria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isopentenyl diphosphate synthesis, unlike humans who use the mevalonate pathway.
- Targeting essential microbial pathways offers a strategy for selective antimicrobial drug development.
Purpose of the Study:
- To characterize the IspD enzyme from Mycobacterium tuberculosis and Mycobacterium smegmatis for structure-based drug discovery.
- To obtain high-resolution crystal structures of IspD in complex with ligands to guide inhibitor design.
Main Methods:
- Engineering and expression of soluble enzyme constructs for Mycobacterium smegmatis and Mycobacterium tuberculosis IspD.
- X-ray crystallography to determine the structures of M. smegmatis IspD with CTP and CMP, and M. tuberculosis IspD with CTP.
- Enzymatic characterization of the M. tuberculosis IspD construct.
Main Results:
- Crystal structures of M. smegmatis IspD (with CTP and CMP) and M. tuberculosis IspD (with CTP) were determined and refined.
- Comparison of structures revealed a conserved core and potential flexibility near the active site.
- Enzymatic characterization of M. tuberculosis IspD was performed.
Conclusions:
- Well-behaved enzyme constructs and their structures are valuable tools for screening potential inhibitors.
- The conserved active site suggests that inhibitors developed against IspD could have broad-spectrum activity against MEP pathway-dependent pathogens.
