Properties of phosphorylated thymidylate synthase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Thymidylate synthase (TS) is crucial for DNA synthesis.
• Endogenous and recombinant TS may undergo phosphorylation.
• Phosphorylation's functional impact on TS is not fully understood.

Purpose of the Study:

• To investigate the phosphorylation of thymidylate synthase (TS).
• To determine the functional consequences of TS phosphorylation.
• To identify specific phosphorylation sites in different cellular contexts.

Main Methods:

• Pro-Q® Diamond Phosphoprotein Gel Stain (PGS) for detecting phosphorylation.
• Recombinant expression of TS in E. coli.
• Enzyme kinetics (Vmax), mRNA binding assays, and translation repression studies.
• Mass spectrometry (ESI-MS/MS, NanoLC-MS/MS) and 31P NMR for site identification.
• Molecular dynamics simulations.

Main Results:

• Phosphorylated recombinant TS showed reduced Vmax(app) and altered mRNA binding and translation repression.
• Phosphorylation sites differed between bacterial (histidine) and mammalian (Ser10, Ser16 in resistant cells) TS.
• Mass spectrometry identified phosphohistidine, phosphoserine, and phosphothreonine residues.
• Molecular dynamics suggested phosphorylation site-dependent effects on catalytic activity.

Conclusions:

• TS phosphorylation significantly modulates enzyme activity and regulatory functions.
• Distinct phosphorylation patterns exist in bacterial versus mammalian systems.
• Further research is needed to fully elucidate TS phosphorylation site(s) and their precise roles.