title: Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/β-lactamase homologue from a metagenomic library.
abstract: Interest in penicillin-binding proteins and β-lactamases (the PBP-βL family) is increasing owing to their biological and clinical significance. In this study, the crystal structure of Est-Y29, a metagenomic homologue of the PBP-βL family, was determined at 1.7 Å resolution. In addition, complex structures of Est-Y29 with 4-nitrophenyl phosphate (4NP) and with diethyl phosphonate (DEP) at 2.0 Å resolution were also elucidated. Structural analyses showed that Est-Y29 is composed of two domains: a β-lactamase fold and an insertion domain. A deep hydrophobic patch between these domains defines a wide active site, and a nucleophilic serine (Ser58) residue is located in a groove defined primarily by hydrophobic residues between the two domains. In addition, three hydrophobic motifs, which make up the substrate-binding site, allow this enzyme to hydrolyze a wide variety of hydrophobic compounds, including fish and olive oils. Furthermore, cross-linked Est-Y29 aggregates (CLEA-Est-Y29) significantly increase the stability of the enzyme as well as its potential for extensive reuse in various deactivating conditions. The structural features of Est-Y29, together with biochemical and biophysical studies, could provide a molecular basis for understanding the properties and regulatory mechanisms of the PBP-βL family and their potential for use in industrial biocatalysts.
authors.affiliation: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors.forename: Tri Duc
authors.initials: TD
authors.lastname: Ngo
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: Bum Han
authors.initials: BH
authors.lastname: Ryu
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: Hansol
authors.initials: H
authors.lastname: Ju
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: Eun Jin
authors.initials: EJ
authors.lastname: Jang
authors.affiliation: Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea.
authors.forename: Kyeong Kyu
authors.initials: KK
authors.lastname: Kim
authors.affiliation: Department of Applied Chemistry and Biological Engineering, College of Engineering, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: T Doohun
authors.initials: TD
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray / Q000379:methods*; D006801:Humans; D056186:Metagenomics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D046915:Penicillin-Binding Proteins; D001618:beta-Lactamases
keywords: metagenomic library; penicillin-binding protein; β-lactamase