Structural Insight Into Aeromonas Hydrophila AHL Synthase AhyI Driving Acyl-ACP Selective Recognition

Background: The gram-negative bacterium Aeromonas hydrophila as the major causative agent of the fish disease motile aeromonad septicemia, uses N-acyl-homoserine lactone quorum sensing signals to coordinate biofilm formation, motility and virulence gene expression in pathogens. Thus, AHL signaling pathway is considered as a therapeutic target against pathogenic A. hydrophila infection. AHL autoinducers biosynthesis in A. hydrophila are specifically catalyzed by an ACP-dependent AHL synthase AhyI using SAM and acyl-ACP as the precursors. Our previously reported AhyI protein heterologously expressed in E. coli strain showed the production characteristics of medium-long chain AHLs, although AhyI was only considered as a short-chain C 4 /C 6 -HSL synthase during the past two decades. Results: : In this study, we carried out the in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs. These resulting data all indicate that C 4 /C 6 -ACP are the native acyl substrates for AhyI against acyl-ACPs with longer linear chains as the non-native acyl donor. In an effort to further understand AhyI acyl-donor substrates preferences, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI), and identified three key hydrophobic residues (I67, F125 and L157) as part of the acyl-chain binding pocket that confer AhyI to selectively recognize native C 4 /C 6 -ACP substrates. The predictions were further supported by computational Ala mutation assay. Conclusions: : Our current studies redefined AhyI protein that is a multiple short- to long-chain AHL molecules synthase with longer acyl-ACPs (C8~C14) as the non-native substrates, and we also theorized that with knowledge of the key residues in AHL signal synthase AhyI to drive acyl-ACP selective recognition.