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

Acta Crystallogr D Biol Crystallogr. 2014 Sep;70(Pt 9):2455-66. doi: 10.1107/S1399004714015272. Epub 2014 Aug 29.

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.

Keywords: metagenomic library; penicillin-binding protein; β-lactamase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Crystallography, X-Ray / methods*
  • Humans
  • Metagenomics*
  • Models, Molecular
  • Molecular Sequence Data
  • Penicillin-Binding Proteins / chemistry*
  • Penicillin-Binding Proteins / metabolism
  • Protein Conformation
  • Sequence Homology, Amino Acid
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • beta-Lactamases / chemistry*
  • beta-Lactamases / metabolism

Substances

  • Penicillin-Binding Proteins
  • beta-Lactamases

Associated data

  • PDB/4P6B
  • PDB/4P85
  • PDB/4P87