Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/β-lactamase homologue from a metagenomic library

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2014-09, Vol.70 (9), p.2455-2466
• Main Authors: Ngo, Tri Duc, Ryu, Bum Han, Ju, Hansol, Jang, Eun Jin, Kim, Kyeong Kyu, Kim, T. Doohun
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; beta-Lactamases - chemistry; beta-Lactamases - metabolism; Biochemistry; Crystallography; Crystallography, X-Ray - methods; Enzymes; Grooves; Homology; Humans; Insertion; metagenomic library; Metagenomics; Models, Molecular; Molecular Sequence Data; penicillin-binding protein; Penicillin-Binding Proteins - chemistry; Penicillin-Binding Proteins - metabolism; Protein Conformation; Proteins; Residues; Sequence Homology, Amino Acid; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; β-lactamase
• ISSN: 1399-0047; 1399-0047
• DOI: 10.1107/S1399004714015272

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.