Despite the conservative DNA sequences among Luxl (Acyl Homoserine Lactones synthase gene) homologs, structure-product relationship of AHL synthase remains to be elucidated. In this study, through degenerate primers and in vitro expression methods, we collected the information of the gene sequences and AHL profiles from nine Luxls among Ensifer adhaerens strains. The chromosome-encoded Luxl (CLux1) distinguished themselves from the plasmid-encoded ones (P-Luxl) not only in the DNA sequences, but also in AHL profiles. The C-Luxls produced only C14-HSL, while the P-Luxls produced predominantly C8-HSL and 3-oxo-C8-HSL. Sequence-product relationship analysis updated our recognition of the role of T140 (Esal) in the 3-oxo-HSL production. Computational calculation based on 3D structures of these Luxls revealed the linear relationship between the chain length and the affinity of amides to AHL synthase in C-LuxI, which was not found in the P-Luxl. We hereby proposed the linear docking affinity as a criterion for the prediction of long-chain AHL production by an AHL synthase. This study extends our understanding on the structure-product relationship of AHL synthases and revealed the distinct chromosome and plasmid origin of this enzyme among E. adhaerens. (C) 2019 Elsevier Inc. All rights reserved.