Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium...

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• The methylerythritol phosphate (MEP) pathway is crucial for isoprenoid biosynthesis in bacteria.
• MEP pathway enzymes are attractive targets for anti-infective drug discovery due to their absence in mammals.
• IspD is the third enzyme in the MEP pathway.

Purpose of the Study:

• To determine the crystal structures of Burkholderia thailandensis IspD (BtIspD) in apo and cytidine triphosphate (CTP)-bound states.
• To characterize the apo structure of Mycobacterium paratuberculosis IspD (MpIspD).
• To provide structural and biophysical data to facilitate the discovery of IspD inhibitors.

Main Methods:

• X-ray crystallography to obtain apo and CTP-bound BtIspD structures, and apo MpIspD structure.
• Circular dichroism to evaluate the melting temperatures (Tm) of MpIspD and BtIspD.
• Isothermal titration calorimetry to assess the binding affinity of CTP for BtIspD.

Main Results:

• CTP binding was observed to stabilize a specific loop (residues 13-19) in BtIspD.
• Moderate Tm values for both MpIspD and BtIspD suggest feasibility of thermal shift assays.
• Binding affinity data for CTP-CTP-BtIspD interaction was quantified.

Conclusions:

• Structural insights into CTP-bound BtIspD provide a basis for rational drug design.
• Biophysical characterization supports the potential for inhibitor screening using thermal shift assays.
• The obtained data are valuable for developing novel anti-infective agents targeting the MEP pathway.