title: Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability.
abstract: OBJECTIVE
  Metallo-β-lactamase (MBL)-based resistance is a threat to the use of most β-lactam antibiotics. Multiple variants of the New Delhi MBL (NDM) have recently been reported. Previous reports indicate that the substitutions affect NDM activity despite being located outside the active site. This study compares the biochemical properties of seven clinically reported NDM variants.
  
  
  METHODS
  NDM variants were generated by site-directed mutagenesis; recombinant proteins were purified to near homogeneity. Thermal stability and secondary structures of the variants were investigated using differential scanning fluorimetry and circular dichroism; kinetic parameters and MIC values were investigated for representative carbapenem, cephalosporin and penicillin substrates.
  
  
  RESULTS
  The substitutions did not affect the overall folds of the NDM variants, within limits of detection; however, differences in thermal stabilities were observed. NDM-8 was the most stable variant with a melting temperature of 72°C compared with 60°C for NDM-1. In contrast to some previous studies, kcat/KM values were similar for carbapenem and penicillin substrates for NDM variants, but differences in kinetics were observed for cephalosporin substrates. Apparent substrate inhibition was observed with nitrocefin for variants containing the M154L substitution. In all cases, cefoxitin and ceftazidime were poorly hydrolysed with kcat/KM values <1 s(-1) μM(-1).
  
  
  CONCLUSIONS
  These results do not define major differences in the catalytic efficiencies of the studied NDM variants and carbapenem or penicillin substrates. Differences in the kinetics of cephalosporin hydrolysis were observed. The results do reveal that the clinically observed substitutions can make substantial differences in thermodynamic stability, suggesting that this may be a factor in MBL evolution.
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Anne
authors.initials: A
authors.lastname: Makena
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Brem
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Inga
authors.initials: I
authors.lastname: Pfeffer
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Rebecca E J
authors.initials: RE
authors.lastname: Geffen
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Wilkins
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Hanna
authors.initials: H
authors.lastname: Tarhonskaya
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
authors.forename: Emily
authors.initials: E
authors.lastname: Flashman
authors.affiliation: Antimicrobial Research Group, Queen Mary University London, London E1 2AT, UK.
authors.forename: Lynette M
authors.initials: LM
authors.lastname: Phee
authors.affiliation: Antimicrobial Research Group, Queen Mary University London, London E1 2AT, UK.
authors.forename: David W
authors.initials: DW
authors.lastname: Wareham
authors.affiliation: Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK christopher.schofield@chem.ox.ac.uk.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1
keywords: antibiotic resistance; carbapenemases; cephalosporins; thermal stability; β-lactams