Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei

Area of Science:

• Biochemistry
• Structural Biology
• Crystallography

Background:

• Lactobacillus casei thymidylate synthase (TS) is a crucial enzyme in nucleotide biosynthesis.
• Understanding TS-substrate interactions is key to developing targeted therapeutics.

Purpose of the Study:

• To elucidate the structural basis of dUMP binding to Lactobacillus casei TS.
• To compare the structures of binary TS-dUMP complexes with previously determined ternary complexes.

Main Methods:

• X-ray crystallography was used to determine the structures of TS-dUMP complexes and phosphate-bound TS.
• Multiple Isomorphous Replacement (MIR) and refinement techniques were employed.

Main Results:

• Two crystal forms of the TS-dUMP complex were solved at 2.55 A resolution, showing dUMP bound equivalently in both active sites.
• dUMP adopts the same conformation and forms identical hydrogen bonds as in ternary complexes.
• Phosphate-bound TS structure at 2.36 A revealed disordered residues (90-119) and confirmed no significant conformational difference with dUMP-bound structures.
• Arg23 was identified as forming a key hydrogen bond with the dUMP phosphate group.

Conclusions:

• Lactobacillus casei TS exhibits a conserved binding mode for its substrate dUMP, irrespective of cofactor presence.
• The enzyme structure is largely conserved between phosphate-bound and dUMP-bound states, with minor crystal packing variations observed.
• Structural insights provide a foundation for understanding TS inhibition and drug design.