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J Antimicrob Chemother. 2015 Feb;70(2):463-9. doi: 10.1093/jac/dku403. Epub 2014 Oct 16.

Biochemical characterization of New Delhi metallo-β-lactamase variants reveals differences in protein stability.

Author information

1
Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK.
2
Antimicrobial Research Group, Queen Mary University London, London E1 2AT, UK.
3
Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, UK christopher.schofield@chem.ox.ac.uk.

Abstract

OBJECTIVES:

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.

KEYWORDS:

antibiotic resistance; carbapenemases; cephalosporins; thermal stability; β-lactams

PMID:
25324420
PMCID:
PMC4291237
DOI:
10.1093/jac/dku403
[Indexed for MEDLINE]
Free PMC Article

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