title: Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis.
abstract: N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KMvalue of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-μM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes.
authors.affiliation: School of Life Sciences, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.|State Key Laboratory for Biocontrol, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.
authors.forename: Xiwen
authors.initials: X
authors.lastname: Liu
authors.affiliation: School of Life Sciences, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.|South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou 510275, P. R. China.
authors.forename: Li-Chuang
authors.initials: LC
authors.lastname: Cao
authors.affiliation: School of Basic Medical Sciences, Anhui Medical University, 81 Meishan Rd., Hefei, Anhui 230032, P. R, China.
authors.forename: Xin-Jiong
authors.initials: XJ
authors.lastname: Fan
authors.affiliation: School of Life Sciences, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.|South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou 510275, P. R. China.
authors.forename: Yu-Huan
authors.initials: YH
authors.lastname: Liu
authors.affiliation: School of Life Sciences, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.|State Key Laboratory for Biocontrol, Sun Yat-sen University, 135 W. Xingang Rd., Guangzhou, Guangdong 510275, P. R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
mesh_terms: D054742:Acyl-Butyrolactones / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D061046:Biological Control Agents / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D053038:Quorum Sensing / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D054742:Acyl-Butyrolactones; D061046:Biological Control Agents; D011994:Recombinant Proteins; D004950:Esterases