Preliminary Study On Crystal Structure Of N-Acyl Homoserine Lactonase AidE

Many pathogenic bacteria such as Pseudomonas aeruginosa and Erwinia carotovora rely on the quorum-sensing (QS) systems to regulate such vital biological functions as antibiotic production, luminescence, motility, plasmid transfer, virulence and biofilm production. QS involves secretion of specific molecules like autoinducers by microorganisms to regulate their intraspecies and interspecied density. QS uses diffusible N-acyl-L-homoserine lactones (AHLs) as intercellular messenger molecules. Lots of organisms contain genes coding for lactonases that hydrolyze AHLs into inactive chemicals, thereby blocking QS.Ochrobactrum sp. AidE, an N-Acyl homoserine lactonase (AHL-lactonases), has an important function in blocking quorum-sensing (QS) systems of many pathogenic bacteria and decreasing pathogenicity. There were no reports about the fuction and structure of AidE. Therefore, efforts were made to solve the crystal structure of AidE. In this study, not only the purification, crystallization and preliminary X-ray analysis of AidE, but also enzyme assay、sequence alignment analysis and phylogenetic analysis have been done. Recombinant AidE was overexpressed and purified to homogeneity by Ni-affinity chromatography, gel filtration chromatography and ion-exchange chromatography resulting in a yield of purified protein with uniform charge、size and molecular weight polymer. Seleno-AidE protein was gained using the improved purification procedures. Furthermore, the broad specificity of AidE to degrade a range of AHLs with different acyl-chain lengths and substituents at the C3 position was found. Sequence alignment analysis suggests that AidE has less than 25% identity to the other known AHL-lactonases. Phylogenetic analysis showed that AidE belongs to the metallo-β-lactamase superfamily and contain a highly conserved HXHXDH metal-binding motif. The initial screening of AidE crystals were carried out using sitting-drop vapour diffusion method and hanging-drop vapour diffusion. Finally, a crystal grown from conditions consisting of 1.575 M ammonium sulfate,0.1 M sodium cacodylate trihydrate pH6.6,0.01 M cobalt (Ⅱ) chloride. The data was indexed, integrated and scaled using HKL2000. The AidE crystal belonged to space group P2221 and assumed ten monomers in the asymmetric unit. Thus, the structure of AidE can not be determined by molecular replacement other than by the phase angle which could be gained by means of the derivative of heavy atoms or selenium. At present, the condition of seleno-AidE crystals was clear, what remains to do is the further optimization for the condition.