Crystal Structure Of Drosophila Melanogaster SCOT

Succinyl-CoA:3-ketoacid Co A transferase (SCOT) is a mitochondrial enzyme in mammalian and plays a crucial role in ketone-body metabolism. Ketoacidosis is in a pathological metabolic state which is marked by high and uncontrollable ketosis. In the circumstance of ketoacidosis, our body cannot control the sythesis of ketone, which leads to the accumulation of ketoacid and pH in blood dramatically decreases. Ketoacidosis is ethal in extreme condition. CoA transferase including in human sapiens, pig heart, sheep kidney, rat brain has been studied and the scientists fond that the transferase worked in the way of ping-pang mechanism. Here, we studied the SCOT in the model organism-Drosophila melanogaster. First of all, we did the secondary structure prediction of Drosophila melanogaster SCOT. Because of the coiled coil structure formed by the N-terminal33amino acids which was disadvantage for crystal, we truncated the protein. We obtained a recombinant plasmid named pET28a-his-TEV-CG1140(34-516aa). Then we transformed the plasmid to Escherichia coli BL21and purified Drosophila melanogaster SCOT(DmSCOT) with the methods of affinity chromatography and size exclusive chromatography. We crystallized the SCOT protein using sitting-drop vapour diffusion method. The structure of DmSCOT was determined after X-ray diffraction data analysis. The resolution of DmSCOT is2.64A and it is belonged to space group P212121, with unit-cell parameters a=76.638, b=101.921, c=122.457□,α=β=γ=90°. DmSCOT preserves the typical SCOT tertiary structure, which adopts an open a/p fold. The DmSCOT monomer consists of N-terminal and C-terminal domains (residues1-247and259-484, respectively), each displaying an α/β/α motif with a central seven-stranded β-sheet. The dimer interface involves63residues from each monomer. The biologically relevant DmSCOT dimmer interface involves hydrophobic interactions and some β-sheet hydrogen bonding. There are12hydrogen bonds (involving residues Glu104, Arg107, Gly110, Alalll, Lys186, Arg189, Asp218, His221, Arg353and His356from both monomers), numerous hydrophobic interactions and some salt bridges stabilizing the dimerization interface. Glu307of DmSCOT can be superposed onto Glu305of pig heart SCOT, which is known to be the conserved glutamate residue in the active site of SCOTs. There are conformational differences between DmSCOT and pig heart SCOT in several loops. The most obvious difference lies in residues125-157of DmSCOT, which contains three extra twisted β-hairpins. Superposition of the DmSCOT structure onto that of pig heart SCOT with bound CoA shows that the residues in the CoA-binding site are highly conserved and that the pocket occupied by the bound CoA in the pig heart SCOT structure is also present in DmSCOT.