Coenzyme A (CoA) is the principal acyl carrier in all living systems. It is estimated that about 4% of all enzymes use CoA or a thioester of CoA as a substrate. The five-step pathway for CoA biosynthesis is common to all organisms. In bacteria, the final step is catalyzed by dephosphocoenzyme A kinase (DPCK), while in mammalian cells, this activity is carried out by a bifunctional enzyme, CoA synthase. DPCK catalyses the phosphorylation of dephosphocoenzyme A (dPCoA) using a phosphate from ATP. The crystallographic structures of the Escherichia coli and Haemophilus influenzae DPCKs have been determined, but there has been no insight into the mode of binding of dPCoA and for the E. coli enzyme, ATP could not be seen. Therefore, we have carried out crystallographic and spectroscopic studies with the E. coli DPCK in order to ascertain the oligomerization state of the enzyme and its interactions with the substrate and cofactor. 
            Co-crystallization techniques were used during crystallization assays with the purpose of obtaining enzyme crystals with ATP and dPCoA bound. After several screenings, crystals with ATP present in the crystallization solutions were obtained in four different crystallization conditions without sulfate ions – that prevented ATP binding - in mother liquor. X-ray diffraction experiments showed that one crystal diffracted to 2.6 Å and belong to the space group C222^₁. Diffraction data were processed and the structure was solved by molecular replacement methods. Structure refinement is being carried out.
             In addition, circular dichroism is being used in denaturation assays aiming to point out if the presence of sulfate, phosphate – that could lead to trimer formation - and ATP in the sample confers thermal stability to the protein. Dynamic light scattering and analytical ultracentrifugation studies are also being performed to determine if the presence of sulfate/phosphate ion drives trimer formation.

Financial Support: CAPES.