Exploring the role of L209 residue in the active site of NDM-1 a metallo-β-lactamase

Marcoccia, Francesca; Leiros, Hanna‐Kirsti S.; Aschi, Massimiliano; Amicosante, Gianfranco; Perilli, Mariagrazia

doi:10.1371/journal.pone.0189686

Cited by 17 publications

(14 citation statements)

References 33 publications

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“…Concerning cefepime, the Y229W mutant exhibited lower k cat value but higher k cat /K m values than those for NDM-1 (Table 2). In the L209F mutant, a drastic reduction in activity toward penicillins, cefazolin, and carbapenems was measured (24). The K m values of L209F-Y229W for penicillins were higher than that of L209F single mutant but comparable to the values calculated for NDM-1.…”

Section: Resultssupporting

confidence: 54%

“…Especially, Y229 forms hydrophobic contacts with several amino acids, including L209, which forms also hydrogen bonds with the aforementioned residue (23). The replacement of L209F causes a drastic reduction in ␤-lactamase activity toward penicillins, cephalosporins, and carbapenems (24). The aim of the present study was to evaluate, by kinetic analysis and molecular dynamic (MD) simulations, the effect of the Y229W substitution on the L209F variant.…”

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confidence: 99%

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Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems

Piccirilli

Brisdelli

Aschi

et al. 2019

Antimicrob Agents Chemother

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The New Delhi metallo-β-lactamase-1 (NDM-1) enzyme is the most common metallo-β-lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on non-active-site residues L209 and Y229 of NDM-1 and to investigate their role in the catalytic mechanism. Specifically, the effect of the Y229W substitution in the L209F variant was evaluated by antimicrobial susceptibility testing, kinetic, and molecular dynamic (MD) studies. The Y229W single mutant and L209F-Y229W double mutant were generated by site-directed mutagenesis. The Km, kcat, and kcat/Km kinetic constants, calculated for the two mutants, were compared with those of (wild-type) NDM-1 and the L209F variant. Compared to the L209F single mutant, the L209F-Y229W double mutant showed a remarkable increase in kcat values of 100-, 240-, 250-, and 420-fold for imipenem, meropenem, benzylpenicillin, and cefepime, respectively. In the L209F-Y229W enzyme, we observed a remarkable increase in kcat/Km of 370-, 140-, and 80-fold for cefepime, meropenem, and cefazolin, respectively. The same behavior was noted using the antimicrobial susceptibility test. MD simulations were carried out on both L209F and L209F-Y229W enzymes complexed with benzylpenicillin, focusing attention on the overall mechanical features and on the differences between the two systems. With respect to the L209F variant, the L209F-Y229W double mutant showed mechanical stabilization of loop 10 and the N-terminal region. In addition, Y229W substitution destabilized both the C-terminal region and the region from residues 149 to 154. The epistatic effect of the Y229W mutation jointly with the stabilization of loop 10 led to a better catalytic efficiency of β-lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies.

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Section: Resultssupporting

confidence: 54%

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confidence: 99%

Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems

Piccirilli

Brisdelli

Aschi

et al. 2019

Antimicrob Agents Chemother

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“…VIM-1 [ 31 ] and L1 [ 32 ] metallo-β-lactamases were obtained from the Clinical Biochemistry and Molecular Biology Laboratories of the University of L’Aquila. The gene bla NDM-1 was cloned without a signal peptide in the pET-24(a) vector using the Nde I and Xho I restriction sites to obtain plasmids pFM-NDM-1 [ 33 , 34 ]. E. coli Novablue competent cells ( endA1 hsdR17 (r K12 − m K12 + ) supE44 thi-1 recA1 gyrA96 relA1 lac F[ proA + B + lacI q Z Δ M15 :Tn 10 ] (Tet R )) were used for the initial cloning as a nonexpression host.…”

Section: Methodsmentioning

confidence: 99%

Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-β-lactamase-1 (NDM-1)

et al. 2020

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New Delhi Metallo-β-lactamase-1 (NDM-1) is the most prevalent type of metallo-β-lactamase, able to hydrolyze almost all antibiotics of the β-lactam group, leading to multidrug-resistant bacteria. To date, there are no clinically relevant inhibitors to fight NDM-1. The use of dromedary polyclonal antibody inhibitors against NDM-1 represents a promising new class of molecules with inhibitory activity. In the current study, immunoreactivities of dromedary Immunoglobulin G (IgG) isotypes containing heavy-chain and conventional antibodies were tested after successful immunization of dromedary using increasing amounts of the recombinant NDM-1 enzyme. Inhibition kinetic assays, performed using a spectrophotometric method with nitrocefin as a reporter substrate, demonstrated that IgG1, IgG2, and IgG3 were able to inhibit not only the hydrolytic activity of NDM-1 but also Verona integron-encoded metallo-β-lactamase (VIM-1) (subclass B1) and L1 metallo-β-lactamase (L1) (subclass B3) with inhibitory concentration (IC50) values ranging from 100 to 0.04 μM. Investigations on the ability of IgG subclasses to reduce the growth of recombinant Escherichia coli BL21(DE3)/codon plus cells containing the recombinant plasmid expressing NDM-1, L1, or VIM-1 showed that the addition of IgGs (4 and 8 mg/L) to the cell culture was unable to restore the susceptibility of carbapenems. Interestingly, IgGs were able to interact with NDM-1, L1, and VIM-1 when tested on the periplasm extract of each cultured strain. The inhibitory concentration was in the micromolar range for all β-lactams tested. A visualization of the 3D structural basis using the three enzyme Protein Data Bank (PDB) files supports preliminarily the recorded inhibition of the three MBLs.

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“…Mutational studies of NDM-1 with L231F resulted in a decreased hydrolytic activity towards carbapenems, penicillins and cephalosporins. 70 The amino acids located at 224 and 233 have been reported to be important in substrate recognition and hydrolysis. 41 , 42 …”

Section: Discussionmentioning

confidence: 99%

Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

Fröhlich

Sørum

Huber

et al. 2020

Journal of Antimicrobial Chemotherapy

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Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

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Exploring the role of L209 residue in the active site of NDM-1 a metallo-β-lactamase

Cited by 17 publications

References 33 publications

Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems

Kinetic Profile and Molecular Dynamic Studies Show that Y229W Substitution in an NDM-1/L209F Variant Restores the Hydrolytic Activity of the Enzyme toward Penicillins, Cephalosporins, and Carbapenems

Inhibitory Potential of Polyclonal Camel Antibodies against New Delhi Metallo-β-lactamase-1 (NDM-1)

Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

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