Identification of the critical role of Tyr-194 in the catalytic activity of a novel N-acyl-homoserine lactonase from marine Bacillus cereus strain Y2.
Enzymatic disruption of quorum-sensing (QS) pathways in pathogenic organisms is a promising anti-infection therapeutic strategy. AHL-lactonase, a potent tool for biocontrol, can hydrolyze QS signal molecule N-acyl-homoserine lactones (AHLs) into inactive products, thereby blocking the QS systems. A marine bacterial isolate Y2, identified as a Bacillus cereus subsp., was found capable of inactivating AHLs. The aiiA gene encoding the AHL-degrading enzyme from bacterial strain Y2 was cloned and expressed in Escherichia coli. The 28-kDa recombinant Y2-AiiA protein was purified and showed strong AHL-degrading activity. Sequence comparisons of Y2-aiiA with known AHL-lactonases revealed high identities in the deduced amino-acid sequences. Functional determination of a potential catalytic residue Tyr-194 of AHL-lactonases was performed by site-directed mutagenesis. As judged by AHL-degrading bioassay, substitution of Tyr-194 with Ala resulted in a dramatic decrease of activity compared with wild-type (WT) recombinant Y2-AiiA, although the expression level of the mutated Y2-AiiA protein was equivalent to that of WT Y2-AiiA. These results suggested that the conserved residue Tyr-194 is critical for catalytic function of the novel AHL-lactonase.
Lu
X
X
Department of Biochemistry, College of Life Science, SunYat-Sen University, Guangzhou, 510275, China.
Yuan
Y
Y
Xue
X-L
XL
Zhang
G-P
GP
Zhou
S-N
SN
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001409:Bacillus cereus / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012623:Seawater / Q000382:microbiology*
D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase