title: Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester.
abstract: Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Jinyeong
authors.initials: J
authors.lastname: Kim
authors.affiliation: College of Pharmacy, Ajou University, Suwon, 16499, South Korea.
authors.forename: Seung-Hyeon
authors.initials: SH
authors.lastname: Seok
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Eunsoo
authors.initials: E
authors.lastname: Hong
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea.
authors.forename: Tae Hyeon
authors.initials: TH
authors.lastname: Yoo
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea. mdseo@ajou.ac.kr.|College of Pharmacy, Ajou University, Suwon, 16499, South Korea. mdseo@ajou.ac.kr.
authors.forename: Min-Duk
authors.initials: MD
authors.lastname: Seo
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon, 16499, South Korea. ywryu@ajou.ac.kr.
authors.forename: Yeonwoo
authors.initials: Y
authors.lastname: Ryu
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004952:Esters; D015870:Gene Expression; D015698:Genomic Library; D007660:Ketoprofen / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D054892:Metagenome; D059013:Microbial Consortia / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012988:Soil Microbiology; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins; D004952:Esters; D011994:Recombinant Proteins; D007660:Ketoprofen; D004950:Esterases
keywords: Crystal structure; Enantioselectivity; Enzyme catalysis; Esterase; X-ray crystallography