Next Article in Journal
Synthesis of Au–Ag Alloy Nanoparticle-Incorporated AgBr Crystals
Next Article in Special Issue
Hydrolysis of Glycosyl Thioimidates by Glycoside Hydrolase Requires Remote Activation for Efficient Activity
Previous Article in Journal
SBA-15 as a Support for Effective Olefin Metathesis Catalysts
Previous Article in Special Issue
Highly Selective Oxidation of 5-Hydroxymethylfurfural to 5-Hydroxymethyl-2-Furancarboxylic Acid by a Robust Whole-Cell Biocatalyst
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Catalysts
Volume 9
Issue 9
10.3390/catal9090744
catalysts-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase

by
Zhihai Liu
1,2,
Alessandra Piccirilli
3,
Dejun Liu
2,
Wan Li
2,
Yang Wang
2 and
Jianzhong Shen
2,*
1
Agricultural Bio-pharmaceutical Laboratory, College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China
2
Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
3
Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell’Aquila, 67100 L’Aquila, Italy
*
Author to whom correspondence should be addressed.
Catalysts 2019, 9(9), 744; https://doi.org/10.3390/catal9090744
Submission received: 25 July 2019 / Revised: 27 August 2019 / Accepted: 28 August 2019 / Published: 2 September 2019
(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)
Browse Figures
Versions Notes

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.

1. Introduction

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. 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. NDM-1 genes are located on plasmids that mediate their dissemination across different bacterial strains [4,5]. However, the clinical success of NDM is also 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].
To date, more than 26 variants differing by a limited number of substitutions have been identified [9]. Previous studies revealed that these substitutions have contributed to NDM-1 to increase the hydrolytic activity toward several β-lactams resulting in an increment of resistance in the host bacteria [10]. Crystal structures showed that NDM-1 presents the typical αβ/βα fold of MBLs [11,12]. In this enzyme, the zinc ions are coordinated by six conserved residues: H120, H122, and H189 for Zn1 (BBL numbering) and D124, C208, and H250 for Zn2 (BBL numbering). The active site is surrounded by Loop 3 (residues 67‒73) and Loop 10 (residues 210‒230), involved in the substrate accommodation [12]. The most frequent substitution in NDM-1 is M154L, found in 11 NDM variants (NDM-4, -5, -7, -8, -12, -13, -15, -17, -19, -20 and -21) [9,13,14,15,16]. Indeed, V88L has been reported in five NDM variants (NDM-5, -17, -20, -21 and -24). Other frequent substitutions are A233V (NDM-6, -15, -19 and -27), D130G (NDM-8 and -14), D130N (NDM-7 and -19), and D95N (NDM-3 and -27) [10]. The single substitutions of M154L and D130G seem to increase the carbapenemase activity in NDM-4 and NDM-14, respectively [17,18]. Moreover, the combination D130G/M154L (NDM-8), reduces the hydrolysis toward carbapenems [19]. The main goal of the study was to evaluate the role of the V88L substitution in the NDM-24 enzyme. The NDM-24 was generated in the laboratory by a site-directed mutagenesis of NDM-1 and the enzyme properties, protein structure, and thermal stability were studied compared with NDM-1 and NDM-5.

2. Results and Discussion

2.1. Phylogenetic Analysis

A phylogenetic analysis of NDM-1 variants was performed in order to classify these enzymes based on their amino acid similarities. Overall, the NDM variants were classified into three major clusters (NDM-1, NDM-4, and NDM-24), two minor clusters (NDM-3 and NDM-6), and two divergent proteins (NDM-14 and NDM-10). As shown in Figure 1, the NDM variants are well categorized. The NDM-1 cluster includes eight variants that showed only one amino acid replacement, except for NDM-18 where an insertion of five amino acids have been found (position 48‒52). In the NDM-4 group, all variants possess the replacement at position 154. In particular, except for NDM-11 containing the M154V substitution, all variants shared M154L. In the NDM-24 group, Valine at position 88 has been replaced by a Leucine (V88L). Concerning the two minor groups, similar characteristics were observed with the D95N and A233V substitution for the NDM-3 and NDM-6 clusters, respectively.

2.2. Functional Study

The NDM-24 variant was obtained by a site-directed mutagenesis by using the NDM-1 as template. All genes (blaNDM-1, blaNDM-5, and blaNDM-24) were cloned into pHSG398, which were controlled by the same promoter and thus the same expression. The E. coli DH5α recombinant strains were used to test susceptibility to a wide panel of β-lactams. As shown in Table 1, the results of the susceptibility test revealed that NDM-1, NDM-5, and NDM-24 confer resistance to most β-lactams with similar MIC values, suggesting that the NDM enzymes were successfully expressed in the host cells. A different behaviour was observed for carbapenems, for which the MIC values were markedly lower than those of penicillins and cephalosporins with the exception of cefepime, as previously reported [15,16,17,18,19,20]. Concerning ertapenem, NDM-24 and NDM-5 showed an increase of the MIC values of the 4- and 8-fold with respect to NDM-1. Based on the data obtained, the V88L substitution enhances resistance toward ertapenem.

2.3. Characteristics of Enzyme Activity

In order to obtain soluble and active enzymes, the recombinant plasmids were successfully expressed in the E. coli BL21 (DE3) cells as described in the methods section. After purification, the enzymes were checked on SDS-PAGE to confirm the solubility and purity (>90%) (Figure S1). The MALDI-TOF mass spectrometry was used to confirm the molecular mass of the three enzymes, which corresponds to 24884,024 Da (Figure S2). To investigate the enzyme activity, the kinetic parameters for NDM-1, NDM-5, and NDM-24 were determined (Table 2).
All the NDM variants of this study were able to hydrolyse all the β-lactams tested. Compared with NDM-1, NDM-24 showed lower Km values for penicillins and ceftazidime whereas for carbapenems they are quite similar. Comparing the kcat values, NDM-24 hydrolyses all β-lactams, except penicillins, better than NDM-1 and NDM-5. In particular, the kcat values of NDM-24 are 2.26-, 1.61-, 2.73-, 2.02-, 2.17-, and 1.75-fold higher than NDM-1 towards penicillin G, ceftazidime, cefepime, imipenem, meropenem, and ertapenem, respectively. This was also confirmed by a slight increase of catalytic efficiency. This result stated the important role of V88L in the substrate hydrolysis. The contribution of V88L is likely that of M154L as demonstrated by the calculation of the kcat/Km rates (Table 2). This was possibly due to differences in the intrinsic properties, such as the enzyme stability, protein expression, and adaptability [21,22,23,24], and nutritional conditions of bacteria in vivo/vitro. Comparing the kcat/Km values of carbapenems, the carbapenemase activity of NDM-5 was similar to NDM-24, but higher than NDM-1. A recent study showed that an increase of the catalytic efficiency (kcat/Km) for meropenem has been ascertained in NDM-5 (V88L and M154L). In NDM-4, which contains only M154L, no significant change has been observed, suggesting that V88L might play a role in enhancing the NDM enzymes activity rather than M154L. Moreover, an increase of the carbapenemase activity was also observed in the evolutionary NDM variants, such as NDM-17 (V88L, M154L, and E170K) and NDM-20 (V88L, M154L, and R270H) [10,15,16].

2.4. Thermal Stability

As previously reported, mutations in the NDM variants can affect the enzymes stability, resulting in changing the persistence lifetime in the bacterial host, and consequent antibiotics resistance [25]. For determining whether the V88L substitution influences the NDM-24 stability property, circular dichroism CD was used to assay the protein stability by recording signal changes. NDM-5 was used as reference to analyze the effect of M154L. Compared with NDM-1 and NDM-5, NDM-24 (59.41 ± 0.06 °C) possessed the lowest melting temperature (Figure 2). Notably, the V88L destabilized effect was compensated by M154L in NDM-5 with a remarkable higher thermal temperature than NDM-24 (69.13 ± 3.6 °C compared to 59.41 ± 0.06 °C). Moreover, NDM-5 showed a higher stability than NDM-1 suggesting the destabilized role of M154L. This was in agreement with a previous document that the M154L mutation would be a turning point for the NDM variants, in which combing M154L with additional substitutions benefit for the NDM enzymes exhibiting increased thermostability [10]. In the NDM-24 group there are four variants (NDM-5, -17, -20, and -21) in which the combination of the V88L and M154L substitutions takes favorable results in terms of the stability and environmental selection.

2.5. Structure Analysis

Previous reports indicated that mutations in the NDM influence the α-helical, β-sheets content, and loop flexibility [26]. For example, the Q123A substitution in NDM-1 leads to a decrease of the α-helical content [27]. To know if the V88L substitution could modify the NDM-24 structure, a secondary structure was determined by the Far-UV CD spectrum. All NDM variants CD spectrum data were fitted and shown in Figure 3. The spectrum signals were superimposable at most wavelengths, and showed characteristics of αβ/βα fold, a typical and conservative protein structure in MBLs [28]. The presence of positive bands at 192 nm and two negative peaks at 208 nm, a minimum peak, and 220 nm, suggesting the dominance of the β-sheets and α-helical content. The major differences were observed in the nearby 192 nm, symbolizing α-helical peak, and 208–220 nm, a α-helical and β-sheets bonds. Overall, the α-helical content was found ranging between 13%–20% in NDM-1, NDM-5, and NDM-24 (Table 3), in agreement with previous reports and the content of the β-sheet was high around 30% [27]. Compared to NDM-1, NDM-24 possesses a higher α-helical content and lower β-sheet content, suggesting that V88L was responsible for the secondary structure content changes of NDM-24. Furthermore, the secondary predicted result (Figure S3) confirmed that the V88L substitution occurred in the β-strand terminal, which may be prematurely terminated, leading to a decrease in the β-sheet content. Kumar et al. claimed that 152A, located in the β-strand, drastically influenced the NDM-5 activity and protein thermolability, by reducing the β-sheet content [26]. Our analysis demonstrates that the emergence of M154L (in NDM-5) caused the α-helical to decrease and the β-sheet to increase relative to NDM-24, while the α-helical and β-sheet content of NDM-5 were between NDM-1 and NDM-24. In addition, the 3D model of NDM-24 (Figure 4) was generated by using NDM-1 (PDB accession: 5N0H, 4RBS, 4HKY, and 4EYL) and NDM-5 (PDB accession: 6MGY, and 4TZE) as a template. Although the residue 88L is away from the active site groove and far to the active loops (Loop 3 and Loop 10), differences in the structure content, stability, and enzyme activity were ascertained. Several studies confirmed that non-activity sites substitution can influence the NDM catalytic efficiency [29], and our results about the V88L substitution support this theory.

3. Material and Methods

3.1. Site-Directed Mutagenesis, Cloning and Expression of NDM Variants

The blaNDM-1 and blaNDM-5 encoding genes were obtained from clinical Escherichia coli strains as previously described [15,16]. Site-directed mutagenesis was performed to generate blaNDM-24 using the pHSG398/NDM-1 plasmid as template and primers listed in Table S1, as previously described [30].
First, the blaNDM-genes were cloned into a pHSG398 vector (TaKaRa Bio, Dalian, China) using the BamHI and XhoI restriction sites. In a second PCR experiment, the blaNDM variants were amplified without a signal peptide introducing the Tobacco Etch Virus (TEV) at the N-terminal sequence. In order to obtain enzymes overexpression, the amplicons were inserted into a pET-28a(+) plasmid. The E. coli DH5α competent cells were used as a non-expression host. E. coli BL21(DE3) was used for enzymes overexpression. The authenticity of recombinant plasmids was verified by PCR and sequencing was with Sanger.

3.2. Antimicrobial Susceptibility Tests

The phenotypic profile has been characterized by a microdilution method using a bacterial inoculum of 5 x 105 CFU/ml according to the Clinical Laboratory and Standards Institute [31,32]. E. coli ATCC25922 was used as a negative control.

3.3. Production and Purification of NDM-1, NDM-5, and NDM-24

NDM-1, NDM-5, and NDM-24 were extracted from 0.5 L of culture of E. coli BL21(DE3)/pETNDM-1, E. coli BL21 (DE3) /pETNDM-5, and E. coli BL21 (DE3)/pETNDM-24, respectively. The cultures were grown at 37 °C to achieve an A600 of 0.5 L, and 0.4 mM of IPTG was added. After addition of the IPTG, the cultures were incubated at 20 °C for 16 h, under aerobic conditions. Thereafter, a cell supernatant containing the soluble NDM protein was obtained from the lytic bacteria by centrifuging at 104 rpm. Proteins purification followed the manufacturer’s instructions (Qiagen, Hilden, Germany) by using the Ni-nitrilotriacetic acid (NTA) agarose. The turbo tobacco etch virus (TEV) protease (Accelagen, San Diego, CA, USA) was used to gain the untagged protein without the His tags. The SDS-PAGE was performed to estimate the NDM purity enzymes. The protein concentration was determined using a BCA Protein Quantification Kit (Vazyme Biotech Co., Ltd, Nanjing, China). The β-lactamase activity was monitored at each purification step using the colour change of nitrocefin 1 mg/mL, a chromogenic cephalosporin, according to the previous report [20].

3.4. Determination of Kinetic Parameters

Steady-state kinetic experiments were performed following the hydrolysis of each substrate at 25 °C in a 50 mM phosphate buffer, pH 7.0 in the presence of 20 µM Zn SO4. The data were collected with a SpectraMax M5 multi-detection microplate reader (Molecular Devices, Sunnyvale, CA, USA) as previously described [33]. Kinetic parameters were determined under initial-rate conditions using the GraphPad Prism® version 5.01 (San Diego, CA, USA) to generate the Michaelis-Menten curves, or by analysing the complete hydrolysis time courses [34,35]. Each kinetic value is the mean of the results of three different measurements. The error was below 5%. NDM-5 was used as a reference to normalize.

3.5. Circular Dichroism and Structure Analysis

The circular dichroism (CD) spectra (185 to 260 nm) were determined with a Chirascan Plus CD spectrophotometer (Applied Photophysics Ltd, Leatherhead, UK) equipped with a Peltier temperature-controlled cell holder, at 25 °C using a 1-mm pathlength cuvette and the Savitzky-Golay filter was explored to the baseline-correct spectra data. Protein concentrations were diluted to 0.05‒0.2 mg/mL with a 5 mM sodium phosphate buffer pH 7.0 [36]. 207 nm spectrum data was used as the baseline to normalize and calibrate data for eliminating minor errors due to different concentrations [37]. The analysis was performed using the CONTINLL and SELCON3 algorithms with reference data sets three and nine, respectively [38]. The super-secondary (tertiary) structures of the proteins were analysed by the CDPro software package, which is available at the CDPro website: https://sites.bmb.colostate.edu/sreeram/CDPro/ [38,39]. To assay the location of the V88L substitution and analyse its effect on the structure, the pharmacophore modeling and screening software program Discovery Studio (version 2018) was employed to generate a three-dimensional (3D) interconnected model of NDM-24 using NDM-1 and NDM-5 as a template, in which reliable data of the crystal structure were collected from the PDB database.

3.6. Thermal Stability Testing

The melting temperature (Tm) was used to show the protein stability. The determination of Tm was performed by recording the CD signal change at 222 nm. Data were collected at a ramp of 1 °C /min with a temperature range from 20‒90 °C. The two-state model using nonlinear regression (Boltzmann) in the OriginPro 9.1.64 (OriginLab, Northampton, MA, USA) was applied to analyse the data. When 50% of the protein melts, the temperature is defined as the Tm, representing thermal stability.

4. Conclusions

Our study explored the NDM-24 biological function and probed the V88L substitution role on the structure, enzyme activity, and stability. In brief, this non-active site change enhances the enzyme activity by increasing the turnover rate, related with an indirect effect on conformation. However, the loss cost caused by V88L significantly decreased the protein stability, and would shorten the persistence lifetime in the cell, so that the resistance to antibiotics hardly exhibits an outstanding elevation for the NDM-24-producing transformants. Meanwhile, alterations in the secondary content, such as lowering the β-sheet, have an interesting role in the NDM instability, being relevant to the V88L substitution occurring in the β-strand. According to previous data, the V88L/M154L combination appears to be favorable in the NDM evolution under an environmental pressure selection [14]. Further analysis about the significance of non-active-site residues will help in the comprehension of the resistance mechanism and broaden insight in the development of inhibitors, such as potential antibiotics candidate by reducing the protein stability lifetime.

Supplementary Materials

The following are available online at https://www.mdpi.com/2073-4344/9/9/744/s1. Figure S1: SDS-PAGE of NDM-24. Lane 1: NDM-24 containing His Tags, Figure S2: Molecular mass spectrometry of NDM-24 estimated by MALDI-TOF, Figure S3: Predicted secondary structure of NDM-24, Lane 2: NDM was cleaved by using Turbo tobacco etch virus (TEV) protease to remove His Tags (Accelagen, San Diego, CA, USA): tagged protein (2a) and untagged protein (2b); Lane 3: untagged protein; Lane M: Marker, Table S1. Oligonucleotides used in this study.

Author Contributions

J.S. designed the study. Z.L., D.L., and W.L. collected the data. Z.L., Y.W., and D.L. analyzed and interpreted the data. Z.L., A.P., D.L., Y.W., and J.S. wrote the report. All authors revised, reviewed and approved the final report.

Funding

The study was supported by grants from the National Key Research and Development Program of China (2018YFD0500300), and the National Natural Science Foundation of China (81861138051 and 81661138002).

Conflicts of Interest

The author declares no conflict of interest.

References

  1. Palzkill, T. Metallo-beta-lactamase structure and function. Ann. N. Y. Acad. Sci. 2013, 1277, 91–104. [Google Scholar] [CrossRef] [PubMed]
  2. Garau, G.; García-Sáez, I.; Bebrone, C.; Anne, C.; Mercuri, P.; Galleni, M.; Frère, J.M.; Dideberg, O. Update of the standard numbering scheme for class B beta-lactamases. Antimicrob. Agents Chemother. 2004, 48, 2347–2349. [Google Scholar] [CrossRef] [PubMed]
  3. Yong, D.; Toleman, M.A.; Giske, C.G.; Cho, H.S.; Sundman, K.; Lee, K.; Walsh, T.R. Characterization of a New Metallo-β-Lactamase Gene, blaNDM-1, and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India. Antimicrob. Agents Chemother. 2009, 53, 5046–5054. [Google Scholar] [CrossRef] [PubMed]
  4. Bonnin, R.A.; Poirel, L.; Carattoli, A.; Nordmann, P. Characterization of an IncFII Plasmid Encoding NDM-1 from Escherichia coli ST131. PLoS ONE 2012, 7, 34752. [Google Scholar] [CrossRef] [PubMed]
  5. Dolejska, M.; Villa, L.; Poirel, L.; Nordmann, P.; Carattoli, A. Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance-nodulation-cell division/multidrug efflux pump. J. Antimicrob. Chemother. 2013, 68, 34–39. [Google Scholar] [CrossRef] [PubMed]
  6. King, D.; Strynadka, N. Crystal structure of New Delhi metallo-betalactamase reveals molecular basis for antibiotic resistance. Protein Sci. 2011, 20, 1484–1491. [Google Scholar] [CrossRef] [PubMed]
  7. Gonzalez, L.J.; Bahr, G.; Nakashige, T.G.; Nolan, E.M.; Bonomo, R.A.; Vila, A.J. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-beta-lactamase. Nat. Chem. Biol. 2016, 12, 516–522. [Google Scholar] [CrossRef] [PubMed]
  8. Gonzalez, L.J.; Bahr, G.; Vila, A.J. Lipidated beta-lactamases: From bench to bedside. Future Microbiol. 2016, 11, 1495–1498. [Google Scholar] [CrossRef]
  9. Khan, A.U.; Maryam, L.; Zarrilli, R. Structure, genetics and worldwide spread of New Delhi metallo-beta-lactamase (NDM): A threat to public health. BMC Microbiol. 2017, 17, 101. [Google Scholar] [CrossRef]
  10. Cheng, Z.; Thomas, P.W.; Ju, L.; Bergstrom, A.; Mason, K.; Clayton, D.; Miller, C.; Bethel, C.R.; Vanpelt, J.; Tierney, D.L.; et al. Evolution of New Delhi metallo-β-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity. J. Biol. Chem. 2018, 293, 12606–12618. [Google Scholar] [CrossRef]
  11. Zhang, H.O.; Hau, Q. Crystal structure of NDM-1 reveals a common β-lactam hydrolysis mechanism. FASEB J. 2011, 25, 2574–2582. [Google Scholar] [CrossRef] [PubMed]
  12. Kim, Y.; Cunningham, M.A.; Mire, J.; Tesar, C.; Sacchettini, J.; Joachimiak, A. NDM-1, the ultimate promiscuous enzyme: Substrate recognition and catalytic mechanism. FASEB J. 2013, 27, 1917–1927. [Google Scholar] [CrossRef] [PubMed]
  13. Liu, L.; Feng, Y.; McNally, A.; Zong, Z. bla NDM-21, a new variant of blaNDM in an Escherichia coli clinical isolate carrying blaCTX-M-55 and rmtB. J. Antimicrob. Chemother. 2018, 73, 2336–2339. [Google Scholar] [CrossRef] [PubMed]
  14. Bahr, G.; Vitor-Horen, L.; Bethel, C.R.; Bonomo, R.A.; González, L.J.; Vila, A.J. Clinical evolution of New Delhi Metallo-β-lactamase (NDM) optimizes resistance under Zn(II) deprivation. Antimicrob. Agents Chemother. 2018, 62, 1817–1849. [Google Scholar] [CrossRef] [PubMed]
  15. Liu, Z.; Li, J.; Wang, X.; Liu, D.; Ke, Y.; Wang, Y.; Shen, J. Novel Variant of New Delhi Metallo-β-lactamase, NDM-20, in Escherichia coli. Front. Microbiol. 2018, 9, 248. [Google Scholar] [CrossRef] [PubMed]
  16. Liu, Z.; Wang, Y.; Walsh, T.R.; Liu, D.; Shen, Z.; Zhang, R.; Yin, W.; Yao, H.; Li, J.; Shen, J. Plasmid-mediated novel blaNDM-17 gene encoding a carbapenemase with enhanced activity in a ST48 Escherichia coli strain. Antimicrob. Agents Chemother. 2017, 61, 2216–2233. [Google Scholar] [CrossRef] [PubMed]
  17. Nordmann, P.; Boulanger, A.E.; Poirel, L. NDM-4 Metallo-β-Lactamase with Increased Carbapenemase Activity from Escherichia coli. Antimicrob. Agents Chemother. 2012, 56, 2184–2186. [Google Scholar] [CrossRef]
  18. Zou, D.; Huang, Y.; Zhao, X.; Liu, W.; Dong, D.; Li, H.; Wang, X.; Huang, S.; Wei, X.; Yan, X.; et al. A Novel New Delhi Metallo-β-Lactamase Variant, NDM-14, Isolated in a Chinese Hospital Possesses Increased Enzymatic Activity against Carbapenems. Antimicrob. Agents Chemother. 2015, 59, 2450–2453. [Google Scholar] [CrossRef]
  19. Tada, T.; Miyoshi-Akiyama, T.; Dahal, R.K.; Sah, M.K.; Ohara, H.; Kirikae, T.; Pokhrel, B.M. NDM-8 Metallo-β-Lactamase in a Multidrug-Resistant Escherichia coli Strain Isolated in Nepal. Antimicrob. Agents Chemother. 2013, 57, 2394–2396. [Google Scholar] [CrossRef]
  20. Tada, T.; Shrestha, B.; Miyoshi-Akiyama, T.; Shimada, K.; Ohara, H.; Kirikae, T.; Pokhrel, B.M. NDM-12, a Novel New Delhi Metallo-β-Lactamase Variant from a Carbapenem-Resistant Escherichia coli Clinical Isolate in Nepal. Antimicrob. Agents Chemother. 2014, 58, 6302–6305. [Google Scholar] [CrossRef]
  21. Ines, S.; Emma, K.; Rudolf, R.; Rumyana, M.; Anne Marie, Q.; Adolf, B. VIM-15 and VIM-16, two new VIM-2-like metallo-beta-lactamases in Pseudomonas aeruginosa isolates from Bulgaria and Germany. Antimicrob. Agents Chemother. 2008, 52, 2977. [Google Scholar]
  22. Patricia, M.; Tomatis, P.E.; Mussi, M.A.; Fernando, P.; Viale, A.M.; Limansky, A.S.; Vila, A.J. Biochemical characterization of metallo-beta-lactamase VIM-11 from a Pseudomonas aeruginosa clinical strain. Antimicrob. Agents Chemother. 2008, 52, 2250. [Google Scholar]
  23. Jose-Manuel, R.M.; Patrice, N.; Nicolas, F.; Laurent, P. VIM-19, a metallo-beta-lactamase with increased carbapenemase activity from Escherichia coli and Klebsiella pneumoniae. Antimicrob. Agents Chemother. 2010, 54, 471–476. [Google Scholar]
  24. Pierre, B.; Carine, B.; Te-Din, H.; Warda, B.; Yves, D.; Ariane, D.; Kurt, H.; Youri, G. Detection and characterization of VIM-31, a new variant of VIM-2 with Tyr224His and His252Arg mutations, in a clinical isolate of Enterobacter cloacae. Antimicrob. Agents Chemother. 2012, 56, 3283. [Google Scholar]
  25. Corbin, B.D.; Seeley, E.H.; Raab, A.; Feldmann, J.; Miller, M.R.; Torres, V.J.; Anderson, K.L.; Dattilo, B.M.; Dunman, P.M.; Gerads, R.; et al. Metal Chelation and Inhibition of Bacterial Growth in Tissue Abscesses. Science 2008, 319, 962–965. [Google Scholar] [CrossRef] [PubMed]
  26. Kumar, G.; Issa, B.; Kar, D.; Biswal, S.; Ghosh, A.S. E152A substitution drastically affects NDM-5 activity. FEMS Microbiol. Lett. 2017, 364. [Google Scholar] [CrossRef] [PubMed]
  27. Ali, A.; Azam, M.W.; Khan, A.U. Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity. Int. J. Biol. Macromol. 2018, 112, 1272–1277. [Google Scholar] [CrossRef] [PubMed]
  28. Carfi, A.; Pares, S.; Duée, E.; Galleni, M.; Duez, C.; Frère, J.M.; Dideberg, O. The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold. EMBO J. 1995, 14, 4914–4921. [Google Scholar] [CrossRef] [PubMed]
  29. Piccirilli, A.; Brisdelli, F.; Aschi, M.; Celenza, G.; Amicosante, G.; Perilli, M. 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. 2019, 63, e02270-18. [Google Scholar] [CrossRef] [Green Version]
  30. Meini, M.-R.; Tomatis, P.E.; Weinreich, D.M.; Vila, A.J. Quantitative Description of a Protein Fitness Landscape Based on Molecular Features. Mol. Boil. Evol. 2015, 32, 1774–1787. [Google Scholar] [CrossRef]
  31. Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard, 11th ed.; CLSI document M07; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
  32. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, 28th ed.; CLSI document M100; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2018. [Google Scholar]
  33. Crowder, M.W.; Walsh, T.R.; Banovic, L.; Pettit, M.; Spencer, J. Overexpression, purification, and characterization of the cloned metallo-beta-lactamase L1 from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 1998, 42, 921. [Google Scholar] [CrossRef] [PubMed]
  34. De Meester, F.; Joris, B.; Reckinger, G. Automated analysis of enzyme inactivation phenomena. Application to β-lactamases and DD-peptidases. Biochem. Pharmacol. 1987, 36, 2393–2403. [Google Scholar] [CrossRef]
  35. Segel, I.H. Biochemical Calculations, 2nd ed.; John Wiley & Sons: New York, NY, USA, 1976; pp. 236–241. [Google Scholar]
  36. Liu, Z.; Zhang, R.; Li, W.; Yang, L.; Liu, D.; Wang, S.; Shen, J.; Wang, Y. Amino acid changes at the VIM-48 C-terminus result in increased carbapenem resistance, enzyme activity and protein stability. J. Antimicrob. Chemother. 2018, 74, 885–893. [Google Scholar] [CrossRef] [PubMed]
  37. Raussens, V.; Ruysschaert, J.-M.; Goormaghtigh, E. Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular dichroism spectra: A new scaling method. Anal. Biochem. 2003, 319, 114–121. [Google Scholar] [CrossRef]
  38. Sreerama, N.; Venyaminov, S.Y.; Woody, R.W. Estimation of the number of α-helical and β-strand segments in proteins using circular dichroism spectroscopy. Protein Sci. 1999, 8, 370–380. [Google Scholar] [CrossRef] [PubMed]
  39. Sreerama, N.; Venyaminov, S.Y.; Woody, R.W. Analysis of Protein Circular Dichroism Spectra Based on the Tertiary Structure Classification. Anal. Biochem. 2001, 299, 271–274. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Catalysts 09 00744 g001 550
Figure 1. New Delhi metallo-β-lactamase-1 (NDM-1) variants phylogenetic analysis. Phylogenetic groups were differently coloured: For example, the NDM-24 cluster is coloured in green.
Figure 1. New Delhi metallo-β-lactamase-1 (NDM-1) variants phylogenetic analysis. Phylogenetic groups were differently coloured: For example, the NDM-24 cluster is coloured in green.
Catalysts 09 00744 g001
Catalysts 09 00744 g002 550
Figure 2. (A) Thermal stability melting curves. (B) Melting temperatures of the NDM enzymes as determined by the circular dichroism analysis: NDM-1, 63.61 ± 0.57 °C; NDM-V88L, 59.41 ± 0.06 °C; and NDM-5, 69.13 ± 3.6 °C. Data are the means of triplicate experiments, with error bars showing the standard deviation (±SD).
Figure 2. (A) Thermal stability melting curves. (B) Melting temperatures of the NDM enzymes as determined by the circular dichroism analysis: NDM-1, 63.61 ± 0.57 °C; NDM-V88L, 59.41 ± 0.06 °C; and NDM-5, 69.13 ± 3.6 °C. Data are the means of triplicate experiments, with error bars showing the standard deviation (±SD).
Catalysts 09 00744 g002
Catalysts 09 00744 g003 550
Figure 3. Normalized circular dichroism (CD) spectra of the NDM enzymes tested. MRE: Mean residue ellipticity.
Figure 3. Normalized circular dichroism (CD) spectra of the NDM enzymes tested. MRE: Mean residue ellipticity.
Catalysts 09 00744 g003
Catalysts 09 00744 g004 550
Figure 4. Cartoon model of NDM-24. To acquire a credible model, the 6 NDM crystal structure (NDM-1(5N0H, 4RBS, 4HKY, and 4EYL) and NDM-5(6MGY, and 4TZE)) were adopted. The residue 88L and active pocket were labelled.
Figure 4. Cartoon model of NDM-24. To acquire a credible model, the 6 NDM crystal structure (NDM-1(5N0H, 4RBS, 4HKY, and 4EYL) and NDM-5(6MGY, and 4TZE)) were adopted. The residue 88L and active pocket were labelled.
Catalysts 09 00744 g004
Table
Table 1. Antimicrobial susceptibility of E. coli BL21(DE3)-DH5α carrying blaNDM-1, blaNDM-5, and blaNDM-24.
Table 1. Antimicrobial susceptibility of E. coli BL21(DE3)-DH5α carrying blaNDM-1, blaNDM-5, and blaNDM-24.
AntibioticMIC (μg/mL)
E. coli
DH5α/pHSG398		E. coli
DH5α/pHSG398-NDM-24		E. coli
DH5α/pHSG398-NDM-1		E. coli
DH5α/pHSG398-NDM-5
Ampicillin2>256>256>256
Penicillin G16>256>256>256
Aztreonam0.0310.0310.0310.031
Cefepime0.031212
Cefotaxime0.062326464
Cefoxitin2128128128
Ceftazidime0.125256256256
Cefazolin2128128256
Ertapenem0.01510.252
Imipenem0.062222
Meropenem0.031112
Table
Table 2. Kinetic parameters of NDM-1, NDM-5, and NDM-24 toward β-lactams ᵅ.
Table 2. Kinetic parameters of NDM-1, NDM-5, and NDM-24 toward β-lactams ᵅ.
Kinetic ParametersEnzymeβ-Lactams b
AMPPENTAGFEPMEMIPMETP
Km(μM)NDM-24638.79 ± 23.86331.30 ± 29.43173.85 ± 9.73318.93 ± 10.86266.24 ± 27.03338.20 ± 24.23125.23 ± 19.08
NDM-11249.98 ± 210.94224.57 ± 13.57213.90 ± 11.01173.55 ± 19.46284.24 ± 7.87234.83 ± 7.44105.54 ± 3.09
NDM-5825.00 ± 0.29315.21 ± 46.6876.45 ± 4.76179.64 ± 12.19275.16 ± 36.87292.97 ± 13.7682.18 ± 3.86
kcat (s−1)NDM-24259.94 ± 23.52179.10 ± 8.1743.13 ± 1.0622.98 ± 0.34151.75 ± 6.69173.16 ± 8.83110.31 ± 7.62
NDM-1254.34 ± 28.9679.28 ± 1.9626.73 ± 0.718.42 ± 0.6375.18 ± 3.4479.81 ± 5.1562.89 ± 1.15
NDM-5346.13 ± 31.30214.13 ± 12.1126.96 ± 0.7513.05 ± 0.24142.48 ± 17.91149.63 ± 2.0283.18 ± 1.67
kcat/Km (μM−1 s−1)NDM-240.410.540.250.0720.570.510.88
NDM-10.200.350.130.0460.260.340.60
NDM-50.400.680.350.0730.520.511.01
kcat/Km (μM−1 s−1) ratio for:NDM-24/NDM-12.001.531.981.492.161.511.46
NDM-5/NDM-241.031.261.421.010.911.001.15
NDM-5/NDM-12.071.922.821.501.961.501.68
a The proteins were initially purified with a His-tag, which was removed after purification. Each kinetic value is the mean of three different measurements; the error was below 5%. b β-lactams: AMP, ampicillin; TAG, ceftazidime; PEN, penicillin G; FEP, cefepime; IPM, imipenem; MEM, meropenem; ETP, ertapenem.
Table
Table 3. Proportions of various secondary structural elements in the NDM-1, NDM-5, and NDM-24 enzymes.
Table 3. Proportions of various secondary structural elements in the NDM-1, NDM-5, and NDM-24 enzymes.
Program Algorithms aStructural Elements bSMP50(9) cSP37(3) cSP29(1) c
NDM-1NDM-5NDM-24NDM-1NDM-5NDM-24NDM-1NDM-5NDM-24
SELCON3H(r)0.0700.0780.0920.0620.0740.0920.0590.0790.087
H(d)0.0850.0880.0890.0810.0880.0890.0780.0870.086
S(r)0.2150.1990.1950.2280.2140.1950.2310.1910.196
S(d)0.1150.1090.1080.1170.1130.1080.1180.1070.108
Trn0.2140.2110.1940.2180.2140.1940.2260.2140.215
Unrd0.2840.2870.2610.2820.2790.2610.2870.2920.285
H(r)+H(d)0.1550.1660.1810.1430.1620.1810.1370.1660.173
S(r)+S(d)0.330.3080.3030.3450.3270.3030.3490.2980.304
CONTINLLH(r)0.0540.0750.0910.0460.0790.0970.0710.0780.093
H(d)0.0790.0920.1010.0890.0950.1030.0920.0960.100
S(r)0.2170.2080.1870.2020.2050.1820.1970.1970.183
S(d)0.1140.1130.1080.1120.1110.1070.1130.1110.107
Trn0.2330.2200.2200.2480.2160.2160.2310.2220.225
Unrd0.3030.2920.2930.3040.2930.2940.2970.2970.292
H(r)+H(d)0.1330.1670.1920.1350.1740.2000.1630.1740.193
S(r)+S(d)0.3310.3210.2950.3140.3160.2890.3100.3080.290
a The CDPro program package was used to analyse the data using two algorithms: CONTINLL and SELCON3. b H(r), regular α-helix; H(d), distorted α-helix; S(r), regular β-strand; S(d), distorted β-strand; Trn, turns; Unrd, unordered. c The reference protein sets (IBasis sets) were adopted.

Share and Cite

MDPI and ACS Style

Liu, Z.; Piccirilli, A.; Liu, D.; Li, W.; Wang, Y.; Shen, J. Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase. Catalysts 2019, 9, 744. https://doi.org/10.3390/catal9090744

AMA Style

Liu Z, Piccirilli A, Liu D, Li W, Wang Y, Shen J. Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase. Catalysts. 2019; 9(9):744. https://doi.org/10.3390/catal9090744

Chicago/Turabian Style

Liu, Zhihai, Alessandra Piccirilli, Dejun Liu, Wan Li, Yang Wang, and Jianzhong Shen. 2019. "Deciphering the Role of V88L Substitution in NDM-24 Metallo-β-Lactamase" Catalysts 9, no. 9: 744. https://doi.org/10.3390/catal9090744

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop