Exploring the role of L209 residue in the active site of NDM-1 a metallo-β-lactamase (original) (raw)
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Antimicrobial Agents and Chemotherapy, 2019
The New Delhi metallo--lactamase-1 (NDM-1) enzyme is the most common metallo--lactamase identified in many Gram-negative bacteria causing severe nosocomial infections. The aim of this study was to focus the attention on nonactive-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 K m , k cat , and k cat /K m 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 k cat 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 k cat /K m 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 -lactams. NDM numbering is used in order to facilitate the comparison with other NDM-1 studies. KEYWORDS NDM, enzyme kinetics, metallo--lactamases M etallo--lactamases (MBLs) are zinc-dependent enzymes which represent the newest generation of broad-spectrum -lactamases able to inactivate almost all classes of -lactams. Based on their protein sequence similarities, three different lineages have been characterized, subclasses B1, B2, and B3 (1). Among subclass B1, New Delhi metallo--lactamase-1 (NDM-1) is one of the most diffused carbapenemases. This feature is due to its ability to hydrolyze all -lactams (with the exception of monobactams), including the last-resort carbapenems. The prevalence of NDM producers was detected mainly in China and India but also in many European countries (2). NDM-1 was first identified in a multidrug-resistant Klebsiella pneumoniae strain isolated from a Swedish patient (3). To date, NDM-1 and its variants have been found in Enterobacteriaceae, including Escherichia coli, Proteus mirabilis, K. pneumoniae, and Citation Piccirilli A, Brisdelli F, Aschi M, Celenza G, Amicosante G, Perilli M. 2019. 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. Antimicrob Agents Chemother 63:e02270-18.
Structure-based computational study of the hydrolysis of New Delhi metallo-β-lactmase-1
Biochemical and Biophysical Research Communications, 2013
New Delhi metallo-b-lactmase-1 (NDM-1) is an enzyme that confers antibiotic resistance to bacteria and is thus a serious threat to human health. Almost all clinically available b-lactam antibiotics can be hydrolyzed by NDM-1. To determine the mechanism behind the wide substrate diversity and strong catalytic ability of NDM-1, we explored the molecular interactions between NDM-1 and different b-lactam antibiotics using computational methods. Molecular dynamics simulations and binding free energy calculations were performed on enzyme-substrate (ES) complex models of NDM-1-Meropenem, NDM-1-Nitrocefin, and NDM-1-Ampicillin constructed by molecular docking. Our computational results suggest that mutant residues Ile35 and Lys216, and active site loop L1 residues 65-73 in NDM-1 play crucial roles in substrate recognition and binding. The results of our study provide new insights into the mechanism behind the enhanced substrate binding and wider substrate spectrum of NDM-1 compared with its homologous enzymes CcrA and IMP-1. These insights may be useful in the discovery and design of specific and potent inhibitors against NDM-1.
Indian Journal of Microbiology, 2016
Emergence of antimicrobial resistance mediated through New Delhi metallo-b-lactamases (NDMs) is a serious therapeutic challenge. Till date, 16 different NDMs have been described. In this study, we report the molecular and structural characteristics of NDM-5 isolated from an Escherichia coli isolate (KOEC3) of bovine origin. Using PCR amplification, cloning and sequencing of full blaNDM gene, we identified the NDM type as NDM-5. Cloning of full gene in E. coli DH5a and subsequent assessment of antibiotic susceptibility of the transformed cells indicated possible role of native promoter in expression blaNDM-5. Translated amino acid sequence had two substitutions (Val88Leu and Met154Leu) compared to NDM-1. Theoretically deduced isoelectric pH of NDM-5 was 5.88 and instability index was 36.99, indicating a stable protein. From the amino acids sequence, a 3D model of the protein was computed. Analysis of the protein structure elucidated zinc coordination and also revealed a large binding cleft and flexible nature of the protein, which might be the reason for broad substrate range. Docking experiments revealed plausible binding poses for five carbapenem drugs in the vicinity of metal ions. In conclusion, results provided possible explanation for wide range of antibiotics catalyzed by NDM-5 and likely interaction modes with five carbapenem drugs.
Antimicrobial agents and chemotherapy, 2015
New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defence mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active site residues in the catalytic mechanism, there is paucity of reports addressing the role of non-active site residues in the structure and function of NDM-1. Here, we investigated the significance of non-active site residue Trp-93 in the structure and function of NDM-1. We cloned blaNDM-1 from E. cloacae clinical strain (EC-15) and introduced Trp93Ala mutation by PCR based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of Trp93Ala mutant were reduced by 4-8 folds against ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem and meropenem. The poor hydrolytic activity of Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of Trp93Ala was reduced by 40-55...
Physical Chemistry Chemical Physics, 2014
New Delhi metallo-b-lactamase-1 (NDM-1) has attracted extensive attention in recent years for its high activity for hydrolyzing almost all b-lactam antibiotics. Like other metallo-b-lactamases (MbLs), NDM-1 features an invariant Asp120 that ligates the zinc ion (ZN2) in the active site. Previous studies showed that substitutions of Asp120 with residues such as Ala, Ser, Asn and Glu dramatically impaired the MbL (BcII, IMP-1, L1) activity, but no consensus about the exact role of Asp120 has reached. Here we constructed D120N mutant of NDM-1 by site-directed mutagenesis. The replacement of Asp120 with Asn, which has much weaker metal ligating capabilities than Asp, severely impaired the lactamase activity without abolishing the ZN2 site. Molecular dynamics simulations suggested that the ZN1-ZN2 distance increased because of mutation, leading to a rearrangement of the active site, including the bridging OH À . Thereby, the Mulliken charges of ZN1 and ZN2 redistributed, especially for ZN2, which might be the major cause of the impaired activity. Reducing the point charges of Asp120 carboxyl oxygens weakened the ionic interactions between Asp120 and ZN2, and the positions of the zinc ions were also changed as a result. It is proposed that Asp120 acts as a strong ZN2 ligand, positioning ZN2 for catalytically important interactions with the substrate, stabilizing the negatively charged amide nitrogen of the hydrolyzed intermediate, and more importantly, orienting the ZN-bound OH À for nucleophilic attacks and protonation. These functions are of general importance for catalyzing b-lactam antibiotics by NDM-1 as well as other MbLs.
Kinetic Studies of Metallo - β - Lactamase NDM - 1
Kinetically metallo-β-lactamases (MBLs) have a broad substrate spectrum profile and are capable of hydrolyzing a range of substrates. In this study we described the steady-state kinetic parameters (K m , V max , K i , and K cat ) of NDM-1 with the Lineweaver-Burk plot. NDM-1 producing Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were used in this study. Results of inhibition constant (K i ) study revealed that K i is directly proportional to MIC, reflecting that low K i value of drugs represent enhanced susceptibility towards microorganisms. The NDM-1 enzyme showed good affinity (low K m , ranged from 25±2 to 148±14 µM) for imipenem plus cilastatin, meropenem, cefoperazone plus sulbactam and piperacillin plus tazobactam whereas Elores, tigecycline and colistin demonstrated lower affinity (high K m ranged from 303±23 to 335±27, 238±18 to 275±21 and 238±22 to 271±22 µM, respectively) for NDM-1 isolated from A. baumannii, E. coli, K. pneumoniae and P. aeruginosa. The catalytic efficiency (K cat /K m ) for Elores ranged 0.054 to 0.065 which is >65% (or > 1.5 fold) lower than those of tigecycline and colistin whose catalytic efficiencies were 0.098 to 0.11 and 0.095 to 0.106 µM -1 S -1 respectively. Low affinity of Elores towards NDM-1 degradation along with very weak catalytic activity (17-22 S -1 ) makes NDM-1 inefficient in hydrolyzing this antibiotic. Further, it is also evident that NDM-1 enzyme had 6 to 7 fold higher catalytic efficiency for imipenem and meropenem and 32 to 61 fold higher catalytic efficiency for cefoperazone plus sulbactam and piperacillin plus tazobactam compared to Elores. These results clearly indicate that β-lactam plus βlactamase inhibitor (BL plus BLI) combinations such as cefoperazone plus sulbactam and piperacillin plus tazobactam are more rapidly hydrolyzed by NDM-1 enzyme followed by imipenem, meropenem, tigecycline and colistin. From the above it can be concluded that Elores, a novel antibiotic adjuvant entity, is the most stable amongst tested antibiotics which are commonly used in Intensive Care Unit (ICU) for treatment of carbapenemresistant Enterobacteriaceae (CRE) infections and is effective against against NDM-1 enzymes. Therefore use of Elores for the infections caused by NDM-1 positive isolated would be the best choice.
PLoS ONE, 2011
The New Delhi Metallo-b-lactamase (NDM-1) gene makes multiple pathogenic microorganisms resistant to all known blactam antibiotics. The rapid emergence of NDM-1 has been linked to mobile plasmids that move between different strains resulting in worldwide dissemination. Biochemical studies revealed that NDM-1 is capable of efficiently hydrolyzing a wide range of b-lactams, including many carbapenems considered as ''last resort'' antibiotics. The crystal structures of metal-free apo-and monozinc forms of NDM-1 presented here revealed an enlarged and flexible active site of class B1 metallo-blactamase. This site is capable of accommodating many b-lactam substrates by having many of the catalytic residues on flexible loops, which explains the observed extended spectrum activity of this zinc dependent b-lactamase. Indeed, five loops contribute ''keg'' residues in the active site including side chains involved in metal binding. Loop 1 in particular, shows conformational flexibility, apparently related to the acceptance and positioning of substrates for cleavage by a zincactivated water molecule.
Antimicrobial Agents and Chemotherapy, 2018
New Delhi metallo--lactamase 1 (NDM-1) is a subclass B1 metallo-lactamase that exhibits a broad spectrum of activity against -lactam antibiotics. Here we report the kinetic study of 6 Q119X variants obtained by site-directed mutagenesis of NDM-1. All Q119X variants were able to hydrolyze carbapenems, penicillins and first-, second-, third-, and fourth-generation cephalosporins very efficiently. In particular, Q119E, Q119Y, Q119V, and Q119K mutants showed improvements in k cat/ K m values for penicillins, compared with NDM-1. The catalytic efficiencies of the Q119K variant for benzylpenicillin and carbenicillin were about 65-and 70-fold higher, respectively, than those of NDM-1. The Q119K and Q119Y enzymes had k cat /K m values for ceftazidime about 25-and 89-fold higher, respectively, than that of NDM-1.
Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-β-lactamase
Archives of Biochemistry and Biophysics, 2015
In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear βlactamase, CphA from Aeromonas hydrophila. According to our results, significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is a ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. This feature of the β-lactamase would be in turn, associated to the different mechanisms that the protein employs to hydrolyze the different antibiotics; i.e. the catalytic promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study will be used to shed some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MβL inhibitors against a broad spectrum of bacterial pathogens.
Metallo-β-Lactamase Inhibitors Inspired on Snapshots from the Catalytic Mechanism
Biomolecules, 2020
β-Lactam antibiotics are the most widely prescribed antibacterial drugs due to their low toxicity and broad spectrum. Their action is counteracted by different resistance mechanisms developed by bacteria. Among them, the most common strategy is the expression of β-lactamases, enzymes that hydrolyze the amide bond present in all β-lactam compounds. There are several inhibitors against serine-β-lactamases (SBLs). Metallo-β-lactamases (MBLs) are Zn(II)-dependent enzymes able to hydrolyze most β-lactam antibiotics, and no clinically useful inhibitors against them have yet been approved. Despite their large structural diversity, MBLs have a common catalytic mechanism with similar reaction species. Here, we describe a number of MBL inhibitors that mimic different species formed during the hydrolysis process: substrate, transition state, intermediate, or product. Recent advances in the development of boron-based and thiol-based inhibitors are discussed in the light of the mechanism of MBLs...