Crystallization And Preliminary X-ray Characterization Of The Dephospho-CoA Kinase And Aminotransferase From Legionella Pneumophila

As an essential cofactor, Coenzyme A (CoA) plays an important role in cellular metabolism. It functions as an acyl group carrier and mediates a wide variety of biochemical reactions in numerous biochemical pathways. In the final two steps of coenzyme A biosynthesis,4’-phosphopantetheine is adenylylated to form Dephosphocoenzyme A (dephospho-CoA) by the enzyme Phosphopantetheine adenylyltransferase (PPAT), followed by phosphorylation of the3’-hydroxyl group of the ribose moiety of dephospho-CoA to form CoA, which is catalyzed by dephospho-CoA kinase (DPCK). In mammalian cells, a single bifunctional enzyme, which contains PPAT and DPCK domains, attributes the final two reactions.DPCK contains the highly conserved P-loop or Walker A sequence motif GXXXXGKT/S (where X represents any residue) responsible for nucleotide binding. This motif preceded by a beta strand and followed by an alpha helix. It interacts with the phosphate groups of the nucleotide.Kinases have to protect the active site from the omnipresent water to avoid becoming ATPases. In many kinase structures, the ligand(s) are thought to induce structural changes to shield their catalytic centers. According to the previous studies of DPCK structures from Escherichia coli, Haemophilus influenzae and Thermus thermophilus HB8, this type of structural change also plays an important role in DPCKs. Thus revealing the domain movement will contribute remarkably to understanding the DPCK phosphorylation mechanism.DPCK from Legionella pneumophila is207amino acids long, with a molecular mass of23.6kDa. It has40%,38%and31%sequence identities with the DPCK proteins from E. coli, H. influenzae and T. thermophilus HB8, respectively. In order to better understand the details of the biological functions and the domain actions of DPCKs, we purified and crystallized L. pneumophila Dephospho-CoA kinase expressed in E. coli and performed preliminary X-ray analysis of the crystal. The protein was crystallized in space group P21212, with unit-cell parameters a=36.29, b=82.20, c=81.80A, using the hanging-drop vapour diffusion method. Diffraction data were collected at100K and the phases were determined using the molecular replacement method.