Specificity and enzyme kinetics of the quorum-quenching N-Acyl homoserine lactone lactonase (AHL-lactonase).
N-Acyl homoserine lactone (AHL) quorum-sensing signals are the vital elements of bacterial quorum-sensing systems, which regulate diverse biological functions, including virulence. The AHL-lactonase, a quorumquenching enzyme encoded by aiiA from Bacillus sp., inactivates AHLs by hydrolyzing the lactone bond to produce corresponding N-acyl homoserines. To characterize the enzyme, the recombinant AHL-lactonase and its four variants were purified. Kinetic and substrate specificity analysis showed that AHL-lactonase had no or little residue activity to non-acyl lactones and noncyclic esters, but displayed strong enzyme activity toward all tested AHLs, varying in length and nature of the substitution at the C3 position of the acyl chain. The data also indicate that the amide group and the ketone at the C1 position of the acyl chain of AHLs could be important structural features in enzyme-substrate interaction. Surprisingly, although carrying a (104)HX- HXDH(109) short sequence identical to the zinc-binding motif of several groups of metallohydrolytic enzymes, AHL-lactonase does not contain or require zinc or other metal ions for enzyme activity. Except for the amino acid residue His-104, which was shown previously to not be required for catalysis, kinetic study and conformational analysis using circular dichroism spectrometry showed that substitution of the other key residues in the motif (His-106, Asp-108, and His-109), as well as His-169 with serine, respectively, caused conformational changes and significant loss of enzyme activity. We conclude that AHL-lactonase is a highly specific enzyme and that the (106)HXDH(109) approximately H(169) of AHL-lactonase represents a novel catalytic motif, which does not rely on zinc or other metal ions for activity.
Wang
Lian-Hui
LH
Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609. lianhui@imcb.nus.edu.sg
Weng
Li-Xing
LX
Dong
Yi-Hu
YH
Zhang
Lian-Hui
LH
D001407:Bacillus; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D004789:Enzyme Activation; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C432003:N-acyl homoserine lactonase