title: Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum.
abstract: A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications.
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon, Republic of Korea.
authors.forename: Chang Woo
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, Korea.
authors.forename: Sena
authors.initials: S
authors.lastname: Kwon
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.
authors.forename: Sun-Ha
authors.initials: SH
authors.lastname: Park
authors.affiliation: Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, Korea.
authors.forename: Boo-Young
authors.initials: BY
authors.lastname: Kim
authors.affiliation: Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, Korea.
authors.forename: Wanki
authors.initials: W
authors.lastname: Yoo
authors.affiliation: Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, Korea.
authors.forename: Bum Han
authors.initials: BH
authors.lastname: Ryu
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon, Republic of Korea.
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.
authors.forename: Seung Chul
authors.initials: SC
authors.lastname: Shin
authors.affiliation: New Drug Development Center, Daegu-Gyeongpuk Medical Innovation Foundation, Daegu, Republic of Korea.
authors.forename: Sunghwan
authors.initials: S
authors.lastname: Kim
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon, Republic of Korea.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of Chemistry, College of Natural Science, Sookmyung Women's University, Seoul, Korea.
authors.forename: T Doohun
authors.initials: TD
authors.lastname: Kim
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon, Republic of Korea.|Department of Polar Sciences, University of Science and Technology, Incheon, Republic of Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry* / Q000378:metabolism; D010463:Peracetic Acid / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D009596:Nitrophenols; D011994:Recombinant Proteins; D004950:Esterases; D010463:Peracetic Acid