Abstract
The New Delhi metallo-β-lactamase-1 (NDM-1) is a typical carbapenemase and plays a crucial role in antibiotic-resistance bacterial infection. Phylogenetic analysis, performed on known NDM-variants, classified NDM enzymes in seven clusters. Three of them include a major number of NDM-variants. In this study, we evaluated the role of the V88L substitution in NDM-24 by kinetical and structural analysis. Functional results showed that V88L did not significantly increase the resistance level in the NDM-24 transformant toward penicillins, cephalosporins, meropenem, and imipenem. Concerning ertapenem, E. coli DH5α/NDM-24 showed a MIC value 4-fold higher than that of E. coli DH5α/NDM-1. The determination of the kcat, Km, and kcat/Km values for NDM-24, compared with NDM-1 and NDM-5, demonstrated an increase of the substrate hydrolysis compared to all the β-lactams tested, except penicillins. The thermostability testing revealed that V88L generated a destabilized effect on NDM-24. The V88L substitution occurred in the β-strand and low β-sheet content in the secondary structure, as evidenced by the CD analysis data. In conclusion, the V88L substitution increases the enzyme activity and decreases the protein stability. This study characterizes the role of the V88L substitution in NDM-24 and provides insight about the NDM variants evolution.
Highlights
Metallo-β-lactamases (MBLs) are a group of enzymes that confer high resistance to most β-lactams.The active site of these enzymes contains one or two zinc ions, that are crucial for catalytic mechanism [1].Based on their amino acid sequences, MBLs have been divided into subclasses B1, B2, and B3 [2].Among subclass B1, the New Delhi metallo-β-lactamase (NDM-1) is one of the most widespread carbapenemase
A phylogenetic analysis of New Delhi metallo-β-lactamase-1 (NDM-1) variants was performed in order to classify these enzymes clusters based (NDM-1, NDM-4, minor (NDM-3 and NDM-6), two divergent on their aminoand acidNDM-24), similarities.two
The blaNDM-1 and blaNDM-5 encoding genes were obtained from clinical Escherichia coli strains as pHSG398/NDM-1 plasmid as template and primers listed in Table S1, as previously described [30]
Summary
Metallo-β-lactamases (MBLs) are a group of enzymes that confer high resistance to most β-lactams.The active site of these enzymes contains one or two zinc ions, that are crucial for catalytic mechanism [1].Based on their amino acid sequences, MBLs have been divided into subclasses B1, B2, and B3 [2].Among subclass B1, the New Delhi metallo-β-lactamase (NDM-1) is one of the most widespread carbapenemase. Metallo-β-lactamases (MBLs) are a group of enzymes that confer high resistance to most β-lactams. The active site of these enzymes contains one or two zinc ions, that are crucial for catalytic mechanism [1]. Based on their amino acid sequences, MBLs have been divided into subclasses B1, B2, and B3 [2]. NDM-1 was first identified in 2008 in a clinical strain of Klebsiella pneumoniae [3]. NDM-1 producing bacteria can hydrolyse all β-lactams (except monobactams), including carbapenems, the “last resort” antibiotics used in clinical therapy. The clinical success of NDM is due to the fact that it is a lipoprotein anchored to the outer membrane, resulting in an unusual stability of NDM-1 and enabling secretion, in Gram-negative bacteria [6,7,8]
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