title: Active site loop conformation regulates promiscuous activity in a lactonase from Geobacillus kaustophilus HTA426.
abstract: Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China; Environmental Science Research and Design Institute of Zhejiang Province, Hangzhou, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China.
authors.forename: Jiao
authors.initials: J
authors.lastname: An
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Guang-Yu
authors.initials: GY
authors.lastname: Yang
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China.
authors.forename: Aixi
authors.initials: A
authors.lastname: Bai
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China.
authors.forename: Baisong
authors.initials: B
authors.lastname: Zheng
authors.affiliation: Laboratory of Structural Biology, School of Medicine, Tsinghua University, Beijing, People's Republic of China.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Lou
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Geng
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Ye
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
authors.forename: Hai-Feng
authors.initials: HF
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China; Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun, People's Republic of China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Institute of Protein Biochemistry, National Research Council, Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D056506:Geobacillus / Q000378:metabolism*; D008968:Molecular Conformation; D016296:Mutagenesis; D044345:Phosphoric Triester Hydrolases / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; C019103:delta-lactonase; C020579:gluconolactonase; D044345:Phosphoric Triester Hydrolases