Next Article in Journal
Characterization of the Joint Microenvironment in Osteoarthritic Joints for In Vitro Strategies for MSC-Based Therapies: A Systematic Review
Previous Article in Journal
Step-by-Step Development of a Recombinase Polymerase Amplification (RPA) Assay for Sex Identification in Papaya
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Applied Biosciences
Volume 3
Issue 4
10.3390/applbiosci3040028
applbiosci-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:
Communication

Phage Anti-Pycsar Proteins Efficiently Degrade β-Lactam Antibiotics

by
Pallav Joshi
1,2,†,
Stefan Krco
1,2,†,
Samuel J. Davis
1,2,
Lachlan Asser
1,
Thomas Brück
3,
Rochelle M. Soo
1,2,
Mikael Bodén
1,
Philip Hugenholtz
1,2,
Liam A. Wilson
4,
Gerhard Schenk
1,5,* and
Marc T. Morris
1,2,*
1
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD 4072, Australia
2
Australian Centre for Ecogenomics, The University of Queensland, Brisbane, QLD 4072, Australia
3
Werner Siemens-Chair of Synthetic Biotechnology, TUM School of Natural Sciences, Technical University of Munich (TUM), 85748 Garching, Germany
4
Chemistry Research Laboratory, Department of Chemistry and the Ineos Oxford Institute for Antimicrobial Research, Oxford University, Oxford OX1 3TA, UK
5
Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work and share first authorship.
Appl. Biosci. 2024, 3(4), 438-449; https://doi.org/10.3390/applbiosci3040028
Submission received: 11 July 2024 / Revised: 21 September 2024 / Accepted: 8 October 2024 / Published: 11 October 2024
Browse Figures
Versions Notes

Abstract

:
Metallo-β-lactamases (MBLs) are members of the structurally conserved but functionally diverse MBL-fold superfamily of metallohydrolases. MBLs are a major concern for global health care as they efficiently inactivate β-lactam antibiotics, including the “last-resort” carbapenems, and no clinically suitable inhibitors are currently available. Increasingly, promiscuous β-lactamase activity is also observed in other members of the superfamily, including from viruses, which represents an underexplored reservoir for future pathways to antibiotic resistance. Here, two such MBL-fold enzymes from Bacillus phages, the cyclic mononucleotide-degrading proteins ApycGoe3 and ApycGrass, are shown to degrade β-lactam substrates efficiently in vitro. In particular, ApycGrass displays a distinct preference for carbapenem substrates with a catalytic efficiency that is within one order of magnitude of the clinically relevant MBL NDM-1. Mutagenesis experiments also demonstrate that the loss of a metal-bridging aspartate residue reduces nuclease activity up to 35-fold but improves carbapenemase activity. In addition, we hypothesise that the oligomeric state significantly influences β-lactamase activity by modifying access to the active site pocket. Together, these observations hint at a possible new avenue of resistance via the spread of phage-borne MBL-fold enzymes with β-lactamase activity.

1. Introduction

Antibiotic resistance has long been recognised as a critical global health concern [1]. The excessive use of antibiotics as preventive and curative measures against infection not only for human health but also in intensive animal husbandry has resulted in a widespread rise in antimicrobial resistance [2]. Amongst the varied mechanisms of antibiotic resistance, the production of β-lactamases, enzymes that break down β-lactam antibiotics, is of particular concern as they are the most widely used (~65% globally) and include “last-resort” antibiotics such as carbapenems (Figure 1A) [1,3]. These β-lactamases are classified based on their structure and mechanism as serine-β-lactamases (SBLs—Ambler classes A, C, and D; see Bush [4]), which utilise a conserved serine residue to hydrolyse the four-membered β-lactam ring, and metallo-β-lactamases (MBLs—Ambler class B), which catalyse the same reaction using a catalytic hydroxide coordinated by one or two zinc ions [3,5]. MBLs are of particular interest as no clinically useful inhibitors are currently available [3,5,6,7]. In contrast, SBLs are targeted by several potent and widely used drugs (e.g., clavulanic acid) [3,8,9].
MBLs are divided into the B1, B2, and B3 subgroups according to their phylogeny, active site residues, metal content, and substrate preferences [3,11,12]. The B1 and B2 subgroups contain the majority of the MBLs of current clinical concern [13], whereas B3 MBLs are primarily associated with environmental microorganisms [11,12,14,15], although some notable B3 members of concern (e.g., AIM-1) have been encountered in clinical environments [16,17]. The three subgroups belong to the MBL-fold protein superfamily [3,11], named after the first enzyme that was shown to contain the characteristic αββα or “MBL”-fold (Figure 1B), the MBL from Bacillus cereus (BcII) [18]. The MBL-fold superfamily is present in all domains of life, and while functionally diverse, they predominantly function as hydrolases. The members include, but are not limited to, endo- and exoribonucleases [19,20,21], phosphatases [22,23,24], lactonases [25,26], glyoxalases [27,28,29], dehalogenases [30], sulfatases [31,32], oxidases [33], and β-lactamases [11,12,34,35,36,37,38]. Recent studies have shown that numerous members of this superfamily exhibit catalytic promiscuity [39,40,41,42,43]. Promiscuous β-lactamase activity is of concern as the corresponding enzymes may represent a cryptic reservoir of antibiotic resistance. For example, two MBL-fold enzymes recovered from deep-sea viral metagenomes, i.e., TupBlac and PNGM-1, are nucleases with promiscuous β-lactamase activity [41,44,45]. While these native nucleases were likely acquired from a bacterial host, and their β-lactamase activity is currently marginal, their presence in a virus raises the question about the possible evolution and dissemination of antimicrobial resistance through horizontal gene transfer back into bacterial hosts.
During a sequence-based survey of B3 MBLs [12], we discovered two distantly related viral MBL-fold enzymes in the genomes of the Bacillus phage vB_Bsum_Goe3 (KY368640) and Grass virus (KF669652), which both belong to the “Bastille-like” viruses within the Spounavirinae subfamily of the Myoviridae [46,47] (note that phages are viruses that infect bacterial cells and coerce them to make more phages rather than cells [48]). These virally-encoded MBL-fold enzymes appear to have been horizontally-acquired from their bacterial hosts, but no closely-related characterised MBL-fold homologs were reported at that time. The recent characterisation of anti-Pycsar enzymes [49] sheds light on the likely in vivo function of these enzymes. The Pyrimidine Cyclase System for Antiphage Resistance (Pycsar) is a recently identified but seemingly widespread bacterial defence mechanism against phages, which is activated by cyclic nucleotide messengers (Figure 1C) [50,51]. In response, Bacillus phages have evolved an anti-Pycsar system by expressing MBL-fold proteins that specifically cleave and thus inactivate cyclic nucleotide messengers [49]. One such anti-Pycsar protein, Apyc1 from the Bacillus phage Bsp38, shares 78.9% and 88.2% sequence identity with the vB_Bsum_Goe3 and Grass virus MBL-fold enzymes [49]. Therefore, we predicted that these MBL proteins should possess nuclease activity (i.e., ApycGoe3 and ApycGrass, respectively). However, since virally encoded β-lactamase activity has been demonstrated in TupBlac and PNGM-1, we evaluated the potential of both enzymatic properties in ApycGoe3 and ApycGrass. We recombinantly expressed the enzymes in an Escherichia coli host, followed by affinity chromatography purification via either an N-terminal maltose binding protein (MBP) or hexahistidine tag. (Note that both systems resulted in the production of soluble, pure enzymes. However, preliminary assays indicated that only the MBP-tagged enzymes displayed significant β-lactamase activity and hence were used for more detailed catalytic characterisation. Any attempts to remove the MBP tag by proteolytic cleavage resulted in the precipitation of the viral MBL proteins. Unless otherwise specified, the terms ApycGoe3 and ApycGrass correspond to the MBP-tagged constructs.) To assess their likely nucleolytic function, the RNase activity of these enzymes was quantitatively assessed using a fluorescence-based activity assay kit (RNase QC Alert kit, Thermo Fisher, Waltham, MA, USA) [44]. To test their β-lactamase activity, we performed continuous in vitro UV-Vis assays following the hydrolysis of representative β-lactam antibiotics.

2. Materials and Methods

The genes for ApycGoe3 (A0A217ER65) and ApycGrass (U5PU04), as well as ApycGoe3 (D178S) and ApycGrass (D161S), were cloned into both pMAL-c5x and pET-24a(+) vectors for subsequent tag-based affinity purification using either Maltose Binding Protein (MBP) or polyhistidine (6xHis) tags, respectively. The expression vectors containing the relevant genes were synthesised commercially (pMAL-c5x: Gene Universal Inc., Newark, DE, USA; pET-24a(+); Twist Biosciences, San Francisco, CA, USA).
The vectors were transferred into chemically competent E. coli Rosetta (DE3) cells via heat shock at 42 °C. Single, isolated colonies were picked and grown in LB medium supplemented with 100 μg/mL of either ampicillin (pMAL-c5x vectors) or kanamycin (pET-24a(+) vectors) for selection. These cultures were used to inoculate larger expression cultures of the LB medium with appropriate antibiotics and were grown under shaking (200 rpm) at 37 °C until they reached an OD600 ≈ 0.6. At this point, protein expression was induced by adding 500 μM IPTG, reducing the temperature to 18 °C, and leaving the culture to grow for a further 12 h. The cells were harvested by centrifugation (20 min, 5000× g), resuspended in lysis buffer (20 mM Tris buffer, pH 8.0, containing 0.15 M NaCl, 150 μM ZnCl2) supplemented with 1 mg/mL lysozyme, 1 mg/mL DNase I, and 1.5 mg/mL EDTA-free protease inhibitor cocktail, and lysed on ice by sonication. Cell debris was removed by centrifugation (40 min, 14,000× g), and the supernatant was loaded onto a 5 mL MBPTrap HP or HisTrap FF column for MBP- and polyhistidine-tagged enzymes, respectively, pre-equilibrated with purification buffer (20 mM Tris buffer, pH 8.0, containing 0.15 M NaCl, 150 μM ZnCl2). The proteins were eluted against 10 mM maltose (MBPTrap HP) or 500 mM imidazole (HisTrap FF). Fractions containing the enzyme (determined by SDS-PAGE analysis; Supplementary Figures S1–S6) were pooled, and excess NaCl was removed via buffer exchange using EconoPac 10DG desalting columns (Bio-Rad, South Granville, NSW, Australia). For the polyhistidine-tagged ApycGoe3 and ApycGrass, the proteins were further purified via size exclusion chromatography by concentrating the proteins and then loading them onto a Hiprep 16/60 Sephacryl S-300 HR column (Cytiva, Marlborough, MA, USA) pre-equilibrated with purification buffer (20 mM Tris, pH 8.0). Both the polyhistidine- and MBP-tagged proteins were stored at 4 °C in 20 mM Tris buffer (pH 8.0).
To quantify hydrolytic activity towards nucleic acids, the activity of the two viral enzymes and two mutant enzymes was assessed against a mixed RNA substrate supplied by a commercially available nuclease activity kit (RNase QC Alert, Thermo Fisher, Waltham, MA, USA), which had been previously used to assess the nuclease activity of TupBlac [44]. The substrate is composed of ribonucleic acids with both a fluorescent probe and a quencher. Upon hydrolysis of the substrate, the fluorescent probe is no longer quenched, which allows for quantitative analysis of the amount of substrate hydrolysed. The assays were conducted in Costar 96-well flat bottom plates with final volumes of 200 μL at 37 °C for 1 h using a Clariostar TRF plate reader instrument (BMG Labtech, Victoria, Australia) at 490/520 nm excitation/emission via the time-resolved fluorescence function and analysed using Reader Control and MARS Data Analysis Software (V6.2, BMG Labtech, Victoria, Australia). Enzyme concentrations of 50 nM were used for all four tested enzymes. The nucleolytic activity of each enzyme was determined by comparison to a standard curve generated with RNase A. The standard curve was produced with 5, 10, 25, and 50 pg of RNase A. Assays were conducted in three independent experiments (triplicates).
The β-lactamase activity of the wild-type and serine mutant enzymes was tested by continuous in vitro UV-Vis assays against the following representative substrates from each major class of β-lactam antibiotics: ampicillin (λ = 235 nm; ε = 900 M−1 cm−1), carbenicillin (λ = 235 nm; ε= 1190 M−1 cm−1), penicillin G (λ = 235 nm; ε = 936 M−1 cm−1), biapenem (λ = 293 nm; ε = 8630 M−1 cm−1), imipenem (λ = 295 nm; ε = 9000 M−1 cm−1), meropenem (λ = 297 nm; ε = 6500 M−1 cm−1), cefaclor (λ = 280 nm; ε = 6410 M−1 cm−1), cefuroxime (λ = 260 nm; ε = 9320 M−1 cm−1), cephalothin (λ = 265 nm; ε = 8790 M−1 cm−1), and aztreonam (λ = 318 nm; ε = 660 M−1 cm−1). The assays were run for 1 min at 25 °C in 50 mM Tris (pH 8.0) using enzyme concentrations of 0.5 μM for ApycGoe3, ApycGoe3 (D178S), and ApycGrass and 0.3–0.5 μM for ApycGrass (D161S). The data were measured in triplicates, and the Michaelis–Meten parameters were determined by nonlinear fitting using Graphpad Prism 9 (Figure S7). All β-lactamase assays were run on an Agilent Cary 60 UV-Vis Spectrophotometer (Santa Clara, CA, USA).

3. Results and Discussion

As predicted, ApycGoe3 and ApycGrass possess significant RNAse activity (see Figure 2 and the details below), supporting the designation of these enzymes as orthologs of Apyc1. Concerningly, ApycGoe3 and ApycGrass also display significant levels of β-lactamase activity (Table 1). For ApycGrass, the catalytic efficiencies (kcat/KM) are greatest for carbapenem substrates (10–15 s−1 mM−1), followed by penicillins (0.5–3 s−1 mM−1), while cephalosporins are less readily hydrolysed (≥0.1 s−1 mM−1). The preference for carbapenems is due to both higher catalytic rates and lower KM values when compared with penicillins. Interestingly, cephalosporins bind more strongly to ApycGrass than the other substrates but are turned over extremely slowly. For ApycGoe3, the catalytic efficiencies are similar for carbapenems (~1 s−1 mM−1) and penicillins (1–3 s−1 mM−1) and are approximately two- to ten-fold higher than for cephalosporins (0.1–0.5 s−1 mM−1). Similar to ApycGrass, the cephalosporins bind significantly tighter than the other substrates but are turned over very slowly. This suggests that while cephalosporins can bind tightly, they are likely oriented in a catalytically non-competent conformation. Consistent with this interpretation is the observation that cephalosporins, but not the other major classes of β-lactams, are competitive inhibitors of other MBL-fold enzymes, namely, SNM1A and SNM1B, two human MBL-fold enzymes involved in DNA repair, with IC50 values in the low μM range [52]. These results suggest that non-competent binding of cephalosporins or inhibitory effects of these β-lactams may be a more common characteristic across the MBL-fold superfamily than previously appreciated and hence warrants further investigation.
It should be noted that ApycGoe3 and ApycGrass are significantly more efficient as β-lactamases than other virally encoded enzymes from the MBL-fold superfamily. For comparison, PNGM-1 has kcat/KM ratios comparable to those of ApycGoe3 and ApycGrass for penicillins and carbapenems (~0.5–5 s−1 mM−1); however, they all appear to bind in catalytically non-competent conformations (leading to kcat values of 10−2–10−4 s−1; Table 1). In particular, the carbapenemase activity of ApycGrass (kcat ~ 1–3 s−1; kcat/KM ~ 10–15 s−1 mM−1) is remarkable, even when compared with “true” MBLs such as the B1 MBL NDM-1, an enzyme that is recognised as a globally distributed clinical concern [1,54] (Table S1). The kcat and kcat/KM values of the NDM-1-catalysed hydrolysis of the carbapenems meropenem (12 s−1, 250 s−1 mM−1) and imipenem (20 s−1, 210 s−1 mM−1) are only one order of magnitude greater than those of ApycGrass. Furthermore, we also note that the turnover rates of both ApycGoe3 and ApycGrass are comparable and, in some cases, superior to those of some SBLs such as the clinically relevant AmpC and OXA-48 (Table S1). This may indicate that these enzymes are predisposed to further evolve into efficient carbapenemases, which could constitute a threat to current treatment options for infections.
Sequence alignments and AlphaFold3 models suggest that both ApycGoe3 and ApycGrass contain the canonical HHH/DHH metal-binding motif observed in B3 MBLs and across the broader MBL-fold superfamily [3,11] (Figure 3 and Figure S8). Notably, they possess the aspartate residue found in the majority of non-β-lactamase MBL-fold hydrolases, which bridges the two metal ions in the active site. This aspartate residue has been suggested to be critical to catalysis in several MBL-fold enzymes [39,55], but it is notably substituted by non-metal-coordinating residues in all true β-lactamase lineages of the superfamily (i.e., in the B1, B2, and B3 MBLs), potentially implying a key role for this residue in the evolution of β-lactamase activity. The replacement of this metal-bridging ligand in class B β-lactamases may facilitate greater structural flexibility (i.e., reduced rigidity), possibly a feature that is important to accommodate a large number of diverse β-lactam substrates. To test this hypothesis, mutant variants of ApycGoe3 and ApycGrass were generated, in which this aspartate residue was replaced by a serine residue, as is found in B3 MBLs [11].
The mutation has, however, minimal impact on the catalytic properties using carbapenems and penicillins as substrates. The preference for carbapenems is retained with kcat/KM values of ~2 s−1 mM−1 and 15 s−1 mM−1 for ApycGoe3 and ApycGrass, respectively (Table 1). For the penicillin substrates, the corresponding values are 0.5–1.5 s−1 mM−1 and 0.5–3 s−1 mM−1. The cephalosporins are again turned over very slowly, but because of the very low KM values in ApycGoe3, they reach kcat/KM values similar to that of the other substrates. While the introduction of the aspartate to serine mutation had a modest impact on the β-lactamase activity of ApycGoe3 and ApycGrass, it reduced their nuclease activity by 5- and 35-fold, respectively. The RNase activity of the native forms of ApycGoe3 and ApycGrass displayed nuclease activities of 4054 mU/min and 10,115 mU/min (equivalent to ~0.015 μM/min/nM and ~0.035 μM/min/nM; Figure 2), while the mutant enzymes displayed activities of 810 mU/min and 289 mU/min, respectively. In comparison, TupBlac, a dual-activity MBL-fold enzyme from a giant mimivirus, was reported to possess a nuclease activity of 0.451 mU/min [44]. It thus appears that both the native and mutant forms of ApycGoe3 and ApycGrass readily degrade β-lactam antibiotics as well as ribonucleic acids and do so more efficiently than comparable viral MBL-fold nucleases, although nuclease activity is greatly diminished in the serine mutant enzymes. Hence, our study demonstrates that while the aspartate residue that is present in the active site of many MBL-fold superfamily members is important for nuclease activity, its contribution to β-lactamase activity is less significant. The loss of this aspartate residue may thus have been a mechanism to enable ancestral MBL-fold hydrolases to accelerate their evolution towards β-lactamase, but other as of yet unknown factors also contribute towards this functional transition.
Interestingly, the hexahistidine-tagged variants of both enzymes display nuclease but no β-lactamase activity (data not shown). A possible explanation for the observed discrepancy in the activity profiles of the different variants may be the impact of the tags on the oligomeric state of these enzymes. We hypothesise that because of its size, the MBP tag prevents the oligomerisation of ApycGoe3 and ApycGrass, while the histidine-tagged variants may form dimers or even higher oligomers. Indeed, structural prediction using AlphaFold3 suggested that the native and polyhistidine-tagged variants form dimers, whereas the enzymes tagged with the larger MBP tag were monomeric (Figure 3). It is noteworthy that most of the highly active “true” MBLs (e.g., NDM-1, AIM-1) are monomeric, while many nucleases such as the RNase Zs are dimeric [19]. In addition, the crystal structure of PNGM-1 (PDB code: 6J4N) suggests that the protein may be tetrameric [45], while structural modelling of TupBlac reveals considerable structural similarity to the dimeric RNase Zs [44]. These results thus raise the possibility that both TupBlac and PNGM-1, as well as multimeric MBL-fold hydrolases more broadly, may be functionalised into more efficient β-lactamases by altering their oligomeric states to monomers, which may enhance the accessibility of the active site for β-lactam substrates.

4. Conclusions

The present study provides evidence that anti-Pycsar proteins from the MBL-fold superfamily are capable of efficient β-lactam hydrolysis, and notably display a preference for the “last-resort” carbapenems. However, their β-lactamase activity may be dependent on their oligomeric state. The metal-bridging aspartate residue present in the large majority of MBL-fold enzymes, but absent in “true” MBLs, appears to have minimal effect on β-lactamase activity (Table 1), but its removal greatly impairs nuclease activity (Figure 2). The evolution of “true” MBLs from an MBL-fold hydrolase precursor could thus have arisen from a concerted effect of structural changes that may alter the oligomeric state and the removal of a metal–ligand that leads to a change in the preferred substrate towards β-lactams. Given that phages can be vectors of horizontal transfer of genes between bacterial hosts, the possible evolution of an efficient β-lactamase from a phage-encoded MBL-fold nuclease, in particular, anti-Pycsar enzymes, could represent a new avenue for the rapid dissemination of antimicrobial resistance and thus pose a significant threat to human health. It is thus very important to gain insight into the environmental conditions (e.g., high bacterial density, stress conditions, presence of mobile genetic elements, nutrient availability) that facilitate or promote horizontal transfer of relevant genes as one avenue to stem the possible spread of antibiotic resistance. As the number of known enzymes within the MBL superfamily with cryptic β-lactamase activity grows, it will also become increasingly more important to design specific inhibition strategies to prevent this activity from providing novel avenues of antibiotic resistance. It is hoped that studies like the present one will stimulate a concerted, global effort in this quest.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/applbiosci3040028/s1: Materials and Methods; Figures S1–S6: SDS-PAGE gels of purified protein samples, Figure S7: Representative Michaelis–Menten curves for reactions catalysed by ApycGoe3, ApycGoe3(D178S), ApycGrass, and ApycGrass (D161S) with relevant substrates. Figure S8: Alignment of metal-binding residues of Apyc orthologs and representatives of the MBL-fold superfamily; Table S1: Catalytic parameters of representative members of each Ambler class of β-lactamases. References [56,57,58,59,60,61,62,63,64,65,66,67,68,69,70] relate to the content in the Supplementary Materials.

Author Contributions

Conceptualisation, S.J.D., L.A.W., R.M.S., M.B., P.H., G.S. and M.T.M.; methodology, P.J., S.K., S.J.D., L.A.W. and M.T.M.; software—validation, T.B., R.M.S., M.B., P.H., G.S. and M.T.M.; formal analysis, P.J., S.K., S.J.D., L.A., L.A.W., and M.T.M.; investigation, P.J., S.K., L.A. and L.A.W.; resources, R.M.S., M.B., P.H., G.S. and M.T.M.; data curation, S.J.D. and L.A.W.; writing—original draft preparation, P.J., S.K., S.J.D. and M.T.M.; writing—review and editing, P.J., S.K., S.J.D., L.A., L.A.W., T.B., R.M.S., M.B., P.H., G.S. and M.T.M.; visualisation, S.J.D., S.K. and M.T.M.; supervision, T.B., R.M.S., M.B., P.H., G.S. and M.T.M.; project administration, P.H., G.S. and M.T.M.; funding acquisition, R.M.S., M.B., P.H. and G.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Health and Medical Research Council from Australia, Ideas Grant ID 2013090.

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of this study; in the collection, analyses, or interpretation of data; in the writing of this manuscript; or in the decision to publish the results.

References

  1. World Health Organization. Antimicrobial Resistance: Global Report on Surveillance; World Health Organization: Geneva, Switzerland, 2014; ISBN 978-92-4-156474-8.
  2. Mann, A.; Nehra, K.; Rana, J.S.; Dahiya, T. Antibiotic Resistance in Agriculture: Perspectives on Upcoming Strategies to Overcome Upsurge in Resistance. Curr. Res. Microb. Sci. 2021, 2, 100030. [Google Scholar] [CrossRef]
  3. Bahr, G.; González, L.J.; Vila, A.J. Metallo-β-Lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design. Chem. Rev. 2021, 121, 7957–8094. [Google Scholar] [CrossRef]
  4. Bush, K. The ABCD’s of β-Lactamase Nomenclature. J. Infect. Chemother. 2013, 19, 549–559. [Google Scholar] [CrossRef]
  5. Brem, J.; Panduwawala, T.; Hansen, J.U.; Hewitt, J.; Liepins, E.; Donets, P.; Espina, L.; Farley, A.J.M.; Shubin, K.; Campillos, G.G.; et al. Imitation of β-Lactam Binding Enables Broad-Spectrum Metallo-β-Lactamase Inhibitors. Nat. Chem. 2022, 14, 15–24. [Google Scholar] [CrossRef]
  6. Arjomandi, O.K.; Hussein, W.M.; Vella, P.; Yusof, Y.; Sidjabat, H.E.; Schenk, G.; McGeary, R.P. Design, Synthesis, and in Vitro and Biological Evaluation of Potent Amino Acid-Derived Thiol Inhibitors of the Metallo-β-Lactamase IMP-1. Eur. J. Med. Chem. 2016, 114, 318–327. [Google Scholar] [CrossRef]
  7. McGeary, R.P.; Tan, D.T.C.; Selleck, C.; Monteiro Pedroso, M.; Sidjabat, H.E.; Schenk, G. Structure-Activity Relationship Study and Optimisation of 2-Aminopyrrole-1-Benzyl-4,5-Diphenyl-1H-Pyrrole-3-Carbonitrile as a Broad Spectrum Metallo-β-Lactamase Inhibitor. Eur. J. Med. Chem. 2017, 137, 351–364. [Google Scholar] [CrossRef]
  8. Neu, H.C.; Fu, K.P. Clavulanic Acid, a Novel Inhibitor of β-Lactamases. Antimicrob. Agents Chemother. 1978, 14, 650–655. [Google Scholar] [CrossRef]
  9. Tooke, C.L.; Hinchliffe, P.; Bragginton, E.C.; Colenso, C.K.; Hirvonen, V.H.A.; Takebayashi, Y.; Spencer, J. β-Lactamases and β-Lactamase Inhibitors in the 21st Century. J. Mol. Biol. 2019, 431, 3472–3500. [Google Scholar] [CrossRef]
  10. Abramson, J.; Adler, J.; Dunger, J.; Evans, R.; Green, T.; Pritzel, A.; Ronneberger, O.; Willmore, L.; Ballard, A.J.; Bambrick, J.; et al. Accurate Structure Prediction of Biomolecular Interactions with AlphaFold 3. Nature 2024, 630, 493–500. [Google Scholar] [CrossRef]
  11. Krco, S.; Davis, S.J.; Joshi, P.; Wilson, L.A.; Monteiro Pedroso, M.; Douw, A.; Schofield, C.J.; Hugenholtz, P.; Schenk, G.; Morris, M.T. Structure, Function, and Evolution of Metallo-β-Lactamases from the B3 Subgroup—Emerging Targets to Combat Antibiotic Resistance. Front. Chem. 2023, 11, 1196073. [Google Scholar] [CrossRef]
  12. Pedroso, M.M.; Waite, D.W.; Melse, O.; Wilson, L.; Mitić, N.; McGeary, R.P.; Antes, I.; Guddat, L.W.; Hugenholtz, P.; Schenk, G. Broad Spectrum Antibiotic-Degrading Metallo-β-Lactamases Are Phylogenetically Diverse. Protein Cell 2020, 11, 613–617. [Google Scholar] [CrossRef]
  13. Bush, K.; Bradford, P.A. β-Lactams and β-Lactamase Inhibitors: An Overview. Cold Spring Harb. Perspect. Med. 2016, 6, a025247. [Google Scholar] [CrossRef] [PubMed]
  14. Pedroso, M.M.; Selleck, C.; Enculescu, C.; Harmer, J.R.; Mitić, N.; Craig, W.R.; Helweh, W.; Hugenholtz, P.; Tyson, G.W.; Tierney, D.L.; et al. Characterization of a Highly Efficient Antibiotic-Degrading Metallo-β-Lactamase Obtained from an Uncultured Member of a Permafrost Community. Metallomics 2017, 9, 1157–1168. [Google Scholar] [CrossRef]
  15. Vella, P.; Miraula, M.; Phelan, E.; Leung, E.W.W.; Ely, F.; Ollis, D.L.; McGeary, R.P.; Schenk, G.; Mitić, N. Identification and Characterization of an Unusual Metallo-β-Lactamase from Serratia Proteamaculans. JBIC J. Biol. Inorg. Chem. 2013, 18, 855–863. [Google Scholar] [CrossRef]
  16. Yong, D.; Toleman Mark, A.; Bell, J.; Ritchie, B.; Pratt, R.; Ryley, H.; Walsh Timothy, R. Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-β-Lactamase Gene, blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia. Antimicrob. Agents Chemother. 2012, 56, 6154–6159. [Google Scholar] [CrossRef]
  17. Zhou, H.; Guo, W.; Zhang, J.; Li, Y.; Zheng, P.; Zhang, H. Draft Genome Sequence of a Metallo-β-Lactamase (Bla(AIM-1))-Producing Klebsiella pneumoniae ST1916 Isolated from a Patient with Chronic Diarrhoea. J. Glob. Antimicrob. Resist. 2019, 16, 165–167. [Google Scholar] [CrossRef]
  18. 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]
  19. Dominski, Z. Nucleases of the Metallo-Beta-Lactamase Family and Their Role in DNA and RNA Metabolism. Crit. Rev. Biochem. Mol. Biol. 2007, 42, 67–93. [Google Scholar] [CrossRef]
  20. Dominski, Z.; Carpousis, A.J.; Clouet-d’Orval, B. Emergence of the β-CASP Ribonucleases: Highly Conserved and Ubiquitous Metallo-Enzymes Involved in Messenger RNA Maturation and Degradation. Biochim. Biophys. Acta BBA-Gene Regul. Mech. 2013, 1829, 532–551. [Google Scholar] [CrossRef]
  21. Pettinati, I.; Brem, J.; Lee, S.Y.; McHugh, P.J.; Schofield, C.J. The Chemical Biology of Human Metallo-β-Lactamase Fold Proteins. Trends BioChem. Sci. 2016, 41, 338–355. [Google Scholar] [CrossRef]
  22. Beaudoin, G.A.W.; Li, Q.; Bruner, S.D.; Hanson, A.D. An Unusual Diphosphatase from the PhnP Family Cleaves Reactive FAD Photoproducts. Biochem. J. 2018, 475, 261–272. [Google Scholar] [CrossRef] [PubMed]
  23. Castillo Villamizar Genis, A.; Funkner, K.; Nacke, H.; Foerster, K.; Daniel, R.; Sawers, G. Functional Metagenomics Reveals a New Catalytic Domain, the Metallo-β-Lactamase Superfamily Domain, Associated with Phytase Activity. mSphere 2019, 4, e00167-19. [Google Scholar] [CrossRef]
  24. Ng, T.K.; Gahan, L.R.; Schenk, G.; Ollis, D.L. Altering the Substrate Specificity of Methyl Parathion Hydrolase with Directed Evolution. Arch. BioChem. Biophys. 2015, 573, 59–68. [Google Scholar] [CrossRef]
  25. Fernandez, F.J.; Garces, F.; López-Estepa, M.; Aguilar, J.; Baldomà, L.; Coll, M.; Badia, J.; Vega, M.C. The UlaG Protein Family Defines Novel Structural and Functional Motifs Grafted on an Ancient RNase Fold. BMC Evol. Biol. 2011, 11, 273. [Google Scholar] [CrossRef]
  26. Miraula, M.; Whitaker, J.J.; Schenk, G.; Mitić, N. β-Lactam Antibiotic-Degrading Enzymes from Non-Pathogenic Marine Organisms: A Potential Threat to Human Health. J. Biol. Inorg. Chem. 2015, 20, 639–651. [Google Scholar] [CrossRef]
  27. Au, S.X.; Dzulkifly, N.S.; Muhd Noor, N.D.; Matsumura, H.; Raja Abdul Rahman, R.N.Z.; Normi, Y.M. Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-β-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence. Int. J. Mol. Sci. 2021, 22, 9377. [Google Scholar] [CrossRef] [PubMed]
  28. Tan, S.H.; Normi, Y.M.; Leow, A.T.C.; Salleh, A.B.; Murad, A.M.A.; Mahadi, N.M.; Rahman, M.B.A. Danger Lurking in the “Unknowns”: Structure-to-Function Studies of Hypothetical Protein Bleg1_2437 from Bacillus lehensis G1 Alkaliphile Revealed an Evolutionary Divergent B3 Metallo-Beta-Lactamase. J. Biochem. 2017, 161, 167–186. [Google Scholar] [CrossRef]
  29. Vašková, J.; Kočan, L.; Vaško, L.; Perjési, P. Glutathione-Related Enzymes and Proteins: A Review. Molecules 2023, 28, 1447. [Google Scholar] [CrossRef]
  30. Wang, G.; Li, R.; Li, S.; Jiang, J. A Novel Hydrolytic Dehalogenase for the Chlorinated Aromatic Compound Chlorothalonil. J. Bacteriol. 2010, 192, 2737–2745. [Google Scholar] [CrossRef]
  31. Barbeyron, T.; Potin, P.; Richard, C.; Collin, O.; Kloareg, B. Arylsulphatase from Alteromonas carrageenovora. Microbiology 1995, 141 Pt. 11, 2897–2904. [Google Scholar] [CrossRef]
  32. Hagelueken, G.; Adams Thorsten, M.; Wiehlmann, L.; Widow, U.; Kolmar, H.; Tümmler, B.; Heinz Dirk, W.; Schubert, W.-D. The Crystal Structure of SdsA1, an Alkylsulfatase from Pseudomonas aeruginosa, Defines a Third Class of Sulfatases. Proc. Natl. Acad. Sci. USA 2006, 103, 7631–7636. [Google Scholar] [CrossRef]
  33. Muok, A.R.; Deng, Y.; Gumerov, V.M.; Chong, J.E.; DeRosa, J.R.; Kurniyati, K.; Coleman, R.E.; Lancaster, K.M.; Li, C.; Zhulin, I.B.; et al. A Di-Iron Protein Recruited as an Fe[II] and Oxygen Sensor for Bacterial Chemotaxis Functions by Stabilizing an Iron-Peroxy Species. Proc. Natl. Acad. Sci. USA 2019, 116, 14955–14960. [Google Scholar] [CrossRef]
  34. Morán-Barrio, J.; Lisa, M.-N.; Larrieux, N.; Drusin, S.I.; Viale, A.M.; Moreno, D.M.; Buschiazzo, A.; Vila, A.J. Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site. Antimicrob. Agents Chemother. 2016, 60, 6013–6022. [Google Scholar] [CrossRef] [PubMed]
  35. Selleck, C.; Larrabee, J.A.; Harmer, J.; Guddat, L.W.; Mitić, N.; Helweh, W.; Ollis, D.L.; Craig, W.R.; Tierney, D.L.; Monteiro Pedroso, M.; et al. AIM-1: An Antibiotic-Degrading Metallohydrolase That Displays Mechanistic Flexibility. Chem.-Eur. J. 2016, 22, 17704–17714. [Google Scholar] [CrossRef]
  36. Wilson, L.A.; Knaven, E.G.; Morris, M.T.; Monteiro Pedroso, M.; Schofield, C.J.; Brück, T.B.; Boden, M.; Waite, D.W.; Hugenholtz, P.; Guddat, L.; et al. Kinetic and Structural Characterization of the First B3 Metallo-β-Lactamase with an Active-Site Glutamic Acid. Antimicrob. Agents Chemother. 2021, 65, e00936-21. [Google Scholar] [CrossRef]
  37. 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]
  38. Garau, G.; Bebrone, C.; Anne, C.; Galleni, M.; Frère, J.-M.; Dideberg, O. A Metallo-β-Lactamase Enzyme in Action: Crystal Structures of the Monozinc Carbapenemase CphA and Its Complex with Biapenem. J. Mol. Biol. 2005, 345, 785–795. [Google Scholar] [CrossRef]
  39. Diene, S.M.; Pinault, L.; Keshri, V.; Armstrong, N.; Khelaifia, S.; Chabrière, E.; Caetano-Anolles, G.; Colson, P.; La Scola, B.; Rolain, J.-M.; et al. Human Metallo-β-Lactamase Enzymes Degrade Penicillin. Sci. Rep. 2019, 9, 12173. [Google Scholar] [CrossRef]
  40. Diene, S.M.; Pinault, L.; Armstrong, N.; Azza, S.; Keshri, V.; Khelaifia, S.; Chabrière, E.; Caetano-Anolles, G.; Rolain, J.-M.; Pontarotti, P.; et al. Dual RNase and β-Lactamase Activity of a Single Enzyme Encoded in Archaea. Life 2020, 10, 280. [Google Scholar] [CrossRef]
  41. Lee, J.H.; Takahashi, M.; Jeon, J.H.; Kang, L.-W.; Seki, M.; Park, K.S.; Hong, M.-K.; Park, Y.S.; Kim, T.Y.; Karim, A.M.; et al. Dual Activity of PNGM-1 Pinpoints the Evolutionary Origin of Subclass B3 Metallo-β-Lactamases: A Molecular and Evolutionary Study. Emerg. Microbes Infect. 2019, 8, 1688–1700. [Google Scholar] [CrossRef]
  42. Miraula, M.; Schenk, G.; Mitić, N. Promiscuous Metallo-β-Lactamases: MIM-1 and MIM-2 May Play an Essential Role in Quorum Sensing Networks. J. Inorg. Biochem. 2016, 162, 366–375. [Google Scholar] [CrossRef] [PubMed]
  43. Perez-Garcia, P.; Kobus, S.; Gertzen, C.G.W.; Hoeppner, A.; Holzscheck, N.; Strunk, C.H.; Huber, H.; Jaeger, K.-E.; Gohlke, H.; Kovacic, F.; et al. A Promiscuous Ancestral Enzyme’s Structure Unveils Protein Variable Regions of the Highly Diverse Metallo-β-Lactamase Family. Commun. Biol. 2021, 4, 132. [Google Scholar] [CrossRef] [PubMed]
  44. Colson, P.; Pinault, L.; Azza, S.; Armstrong, N.; Chabriere, E.; La Scola, B.; Pontarotti, P.; Raoult, D. A Protein of the Metallo-Hydrolase/Oxidoreductase Superfamily with Both Beta-Lactamase and Ribonuclease Activity Is Linked with Translation in Giant Viruses. Sci. Rep. 2020, 10, 21685. [Google Scholar] [CrossRef]
  45. Park, K.S.; Hong, M.-K.; Jeon, J.W.; Kim, J.H.; Jeon, J.H.; Lee, J.H.; Kim, T.Y.; Karim, A.M.; Malik, S.K.; Kang, L.-W.; et al. The Novel Metallo-β-Lactamase PNGM-1 from a Deep-Sea Sediment Metagenome: Crystallization and X-Ray Crystallographic Analysis. Acta Crystallogr. Sect. F Struct. Biol. Commun. 2018, 74, 644–649. [Google Scholar] [CrossRef]
  46. Miller, S.Y.; Colquhoun, J.M.; Perl, A.L.; Chamakura, K.R.; Kuty Everett, G.F. Complete Genome of Bacillus subtilis Myophage Grass. Genome Announc. 2013, 1, e00857-13. [Google Scholar] [CrossRef] [PubMed]
  47. Willms, I.M.; Hoppert, M.; Hertel, R. Characterization of Bacillus subtilis Viruses vB_BsuM-Goe2 and vB_BsuM-Goe3. Viruses 2017, 9, 146. [Google Scholar] [CrossRef]
  48. Youle, M.; Pantéa, L. Thinking Like a Phage: The Genius of the Viruses That Infect Bacteria and Archaea; Wholon: San Diego, CA, USA, 2017; pp. 1–4. [Google Scholar]
  49. Hobbs, S.J.; Wein, T.; Lu, A.; Morehouse, B.R.; Schnabel, J.; Leavitt, A.; Yirmiya, E.; Sorek, R.; Kranzusch, P.J. Phage Anti-CBASS and Anti-Pycsar Nucleases Subvert Bacterial Immunity. Nature 2022, 605, 522–526. [Google Scholar] [CrossRef]
  50. Cohen, D.; Melamed, S.; Millman, A.; Shulman, G.; Oppenheimer-Shaanan, Y.; Kacen, A.; Doron, S.; Amitai, G.; Sorek, R. Cyclic GMP–AMP Signalling Protects Bacteria against Viral Infection. Nature 2019, 574, 691–695. [Google Scholar] [CrossRef] [PubMed]
  51. Tal, N.; Morehouse, B.R.; Millman, A.; Stokar-Avihail, A.; Avraham, C.; Fedorenko, T.; Yirmiya, E.; Herbst, E.; Brandis, A.; Mehlman, T.; et al. Cyclic CMP and Cyclic UMP Mediate Bacterial Immunity against Phages. Cell 2021, 184, 5728–5739.e16. [Google Scholar] [CrossRef]
  52. Lee, S.Y.; Brem, J.; Pettinati, I.; Claridge, T.D.W.; Gileadi, O.; Schofield, C.J.; McHugh, P.J. Cephalosporins Inhibit Human Metallo β-Lactamase Fold DNA Repair Nucleases SNM1A and SNM1B/Apollo. Chem. Commun. 2016, 52, 6727–6730. [Google Scholar] [CrossRef]
  53. Lee, J.H.; Takahashi, M.; Jeon, J.H.; Kang, L.-W.; Seki, M.; Park, K.S.; Hong, M.-K.; Park, Y.S.; Kim, T.Y.; Karim, A.M.; et al. Dual Activity of PNGM-1, a Metallo-β-Lactamase and tRNase Z, Pinpoints the Evolutionary Origin of Subclass B3 Metallo-β-Lactamases. bioRxiv 2019, 8, 575373. [Google Scholar] [CrossRef]
  54. Khan, A.U.; Maryam, L.; Zarrilli, R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-Lactamase (NDM): A Threat to Public Health. BMC Microbiol. 2017, 17, 101. [Google Scholar] [CrossRef] [PubMed]
  55. Malgapo, M.I.P.; Safadi, J.M.; Linder, M.E. Metallo-β-Lactamase Domain-Containing Protein 2 Is S-Palmitoylated and Exhibits Acyl-CoA Hydrolase Activity. J. Biol. Chem. 2021, 296, 100106. [Google Scholar] [CrossRef]
  56. Katoh, K.; Standley, D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013, 30, 772–780. [Google Scholar] [CrossRef]
  57. Rozewicki, J.; Li, S.; Amada, K.M.; Standley, D.M.; Katoh, K. MAFFT-DASH: Integrated Protein Sequence and Structural Alignment. Nucleic Acids Res. 2019, 47, W5–W10. [Google Scholar] [CrossRef] [PubMed]
  58. Bottoni, C.; Perilli, M.; Marcoccia, F.; Piccirilli, A.; Pellegrini, C.; Colapietro, M.; Sabatini, A.; Celenza, G.; Kerff, F.; Amicosante, G.; et al. Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems. Antimicrob. Agents Chemother. 2016, 60, 3123–3126. [Google Scholar] [CrossRef]
  59. Horsfall, L.E.; Izougarhane, Y.; Lassaux, P.; Selevsek, N.; Liénard, B.M.R.; Poirel, L.; Kupper, M.B.; Hoffmann, K.M.; Frère, J.-M.; Galleni, M.; et al. Broad Antibiotic Resistance Profile of the Subclass B3 Metallo-β-Lactamase GOB-1, a Di-Zinc Enzyme. FEBS J. 2011, 278, 1252–1263. [Google Scholar] [CrossRef]
  60. Segatore, B.; Massidda, O.; Satta, G.; Setacci, D.; Amicosante, G. High Specificity of cphA-Encoded Metallo-Beta-Lactamase from Aeromonas hydrophila AE036 for Carbapenems and Its Contribution to Beta-Lactam Resistance. Antimicrob. Agents Chemother. 1993, 37, 1324–1328. [Google Scholar] [CrossRef]
  61. Bebrone, C.; Anne, C.; De Vriendt, K.; Devreese, B.; Rossolini, G.M.; Van Beeumen, J.; Frère, J.-M.; Galleni, M. Dramatic Broadening of the Substrate Profile of the Aeromonas Hydrophila CphA Metallo-β-Lactamase by Site-Directed Mutagenesis. J. Biol. Chem. 2005, 280, 28195–28202. [Google Scholar] [CrossRef]
  62. Venkatachalam, K.V.; Huang, W.; LaRocco, M.; Palzkill, T. Characterization of TEM-1 Beta-Lactamase Mutants from Positions 238 to 241 with Increased Catalytic Efficiency for Ceftazidime. J. Biol. Chem. 1994, 269, 23444–23450. [Google Scholar] [CrossRef]
  63. De Wals, P.-Y.; Doucet, N.; Pelletier, J.N. High Tolerance to Simultaneous Active-Site Mutations in TEM-1 β-Lactamase: Distinct Mutational Paths Provide More Generalized β-Lactam Recognition. Protein Sci. 2009, 18, 147–160. [Google Scholar] [CrossRef] [PubMed]
  64. Poirel, L.; Héritier, C.; Tolün, V.; Nordmann, P. Emergence of Oxacillinase-Mediated Resistance to Imipenem in Klebsiella pneumoniae. Antimicrob. Agents Chemother. 2004, 48, 15–22. [Google Scholar] [CrossRef]
  65. Robin, F.; Delmas, J.; Machado, E.; Bouchon, B.; Peixe, L.; Bonnet, R. Characterization of the Novel CMT Enzyme TEM-154. Antimicrob. Agents Chemother. 2011, 55, 1262–1265. [Google Scholar] [CrossRef]
  66. Marcoccia, F.; Leiros, H.-K.S.; Aschi, M.; Amicosante, G.; Perilli, M. Exploring the Role of L209 Residue in the Active Site of NDM-1 a Metallo-β-Lactamase. PLoS ONE 2018, 13, e0189686. Available online: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189686 (accessed on 1 July 2024). [CrossRef]
  67. Chiou, J.; Cheng, Q.; Shum, P.T.; Wong, M.H.; Chan, E.W.; Chen, S. Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity. Int. J. Mol. Sci. 2021, 22, 11480. [Google Scholar] [CrossRef]
  68. Mammeri, H.; Galleni, M.; Nordmann, P. Role of the Ser-287-Asn Replacement in the Hydrolysis Spectrum Extension of AmpC β-Lactamases in Escherichia coli. Antimicrob. Agents Chemother. 2009, 53, 323–326. [Google Scholar] [CrossRef] [PubMed]
  69. Mazzariol, A.; Cornaglia, G.; Nikaido, H. Contributions of the AmpC β-Lactamase and the AcrAB Multidrug Efflux System in Intrinsic Resistance of Escherichia coli K-12 to β-Lactams. Antimicrob. Agents Chemother. 2000, 44, 1387–1390. [Google Scholar] [CrossRef]
  70. Lenfant, F.; Petit, A.; Labia, R.; Maveyraud, L.; Samama, J.-P.; Masson, J.-M. Site-Directed Mutagenesis of β-Lactamase TEM-1. Eur. J. Biochem. 1993, 217, 939–946. [Google Scholar] [CrossRef]
Applbiosci 03 00028 g001
Figure 1. (A) Chemical structure of the backbones of the four major β-lactam antibiotic classes. (B) Overall structure of ApycGoe3 highlighting the αββα fold. The colouring is a gradient from blue (N-terminal) to red (C-terminal). The two grey spheres represent Zn(II) ions. The structural model was predicted using AlphaFold3 [10]. (C) Chemical structures of the following cyclic nucleotides: cyclic cytidine monophosphate (cCMP) and cyclic uridine monophosphate (cUMP).
Figure 1. (A) Chemical structure of the backbones of the four major β-lactam antibiotic classes. (B) Overall structure of ApycGoe3 highlighting the αββα fold. The colouring is a gradient from blue (N-terminal) to red (C-terminal). The two grey spheres represent Zn(II) ions. The structural model was predicted using AlphaFold3 [10]. (C) Chemical structures of the following cyclic nucleotides: cyclic cytidine monophosphate (cCMP) and cyclic uridine monophosphate (cUMP).
Applbiosci 03 00028 g001
Applbiosci 03 00028 g002
Figure 2. Nuclease activity of ApycGrass, ApycGrass (D161S), ApycGoe3, and ApycGoe3 (D178S) using a mixed ribonucleic acid substrate tagged with a fluorescent probe from the RNase QC Alert kit (Thermo Fisher, Waltham, MA, USA). Activity was obtained by comparison to a standard curve generated with an RNase A positive control.
Figure 2. Nuclease activity of ApycGrass, ApycGrass (D161S), ApycGoe3, and ApycGoe3 (D178S) using a mixed ribonucleic acid substrate tagged with a fluorescent probe from the RNase QC Alert kit (Thermo Fisher, Waltham, MA, USA). Activity was obtained by comparison to a standard curve generated with an RNase A positive control.
Applbiosci 03 00028 g002
Applbiosci 03 00028 g003
Figure 3. (A) Active site of ApycGoe3 showing the aspartate-to-serine mutation observed in ApycGoe3 (D178S), which removes a coordination point for each metal (Zn2+) ion. (B) Overall structure of the polyhistidine-tagged ApycGoe3 dimer. One monomer is coloured in a gradient from blue (N-terminus) to red (C-terminus), and the other is coloured grey. (C) Overall structure of the MBP-tagged ApycGoe3 monomer. The ApycGoe3 portion of the fusion protein is coloured in a gradient from blue (N-terminus) to red (C-terminus), whereas the MBP tag is coloured grey. All structural models were predicted using AlphaFold 3 [10].
Figure 3. (A) Active site of ApycGoe3 showing the aspartate-to-serine mutation observed in ApycGoe3 (D178S), which removes a coordination point for each metal (Zn2+) ion. (B) Overall structure of the polyhistidine-tagged ApycGoe3 dimer. One monomer is coloured in a gradient from blue (N-terminus) to red (C-terminus), and the other is coloured grey. (C) Overall structure of the MBP-tagged ApycGoe3 monomer. The ApycGoe3 portion of the fusion protein is coloured in a gradient from blue (N-terminus) to red (C-terminus), whereas the MBP tag is coloured grey. All structural models were predicted using AlphaFold 3 [10].
Applbiosci 03 00028 g003
Table 1. Catalytic parameters of the MBP-tagged wild-type and mutant forms of the virally encoded ApycGoe3 and ApycGrass recorded with representative substrates from all major classes of β-lactam antibiotics. Units of kcat, KM, and kcat/KM are s−1, μM, and s−1 mM−1|N.H.—no hydrolytic activity detected. a Lee et al. [53]. Errors are shown as standard errors from the mean using triplicate measurements.
Table 1. Catalytic parameters of the MBP-tagged wild-type and mutant forms of the virally encoded ApycGoe3 and ApycGrass recorded with representative substrates from all major classes of β-lactam antibiotics. Units of kcat, KM, and kcat/KM are s−1, μM, and s−1 mM−1|N.H.—no hydrolytic activity detected. a Lee et al. [53]. Errors are shown as standard errors from the mean using triplicate measurements.
ApycGoe3ApycGoe3 (D178S)ApycGrassApycGrass (D161S)PNGM-1 a
SubstratekcatKMkcat/KMkcatKMkcat/KMkcatKMkcat/KMkcatKMkcat/KMkcatKMkcat/KM
Penicillins
Penicillin G0.24 ± 4 × 10−2230 ± 471.040.45 ± 5 × 10−2751 ± 1580.600.67 ± 6 × 10−2231 ± 552.900.48 ± 3 × 10−2180 ± 352.707.5 × 10−2164.7
Ampicillin1.8 ± 0.1663 ± 942.670.72 ± 7 × 10−2526 ± 911.370.54 ± 7 × 10−2418 ± 1021.290.39 ± 5 × 10−2891 ± 1770.442.7 × 10−2151.8
Carbenicillin0.32 ± 2 × 10−2205 ± 391.560.13 ± 9 × 10−3157 ± 290.830.15 ± 2 × 10−2330 ± 820.450.24 ± 4 × 10−2462 ± 1480.52---
Carbapenems
Meropenem0.22 ± 2 × 10−2215 ± 571.020.43 ± 4 × 10−2160 ± 322.691.1 ± 9 × 10−298 ± 2611.24.4 ± 0.7287 ± 8315.38.0 × 10−420.42
Imipenem0.27 ± 3 × 10−2436 ± 330.62---3.1 ± 0.42200 ± 5415.54.2 ± 0.6253 ± 5616.61.1 × 10−320.55
Cephalosporins
Cefuroxime8.6 × 10−3 ± 4 × 10−418 ± 30.488.3 × 10−3 ± 3 × 10−43.5 ± 0.52.375.3 × 10−3 ± 2 × 10−444 ± 90.126.0 × 10−3 ± 9 × 10−4142 ± 430.042---
Cephalothin3.0 × 10−3 ± 2 × 10−419 ± 60.167.0 × 10−3 ± 5 × 10−423 ± 40.305.6 × 10−3 ± 4 × 10−451 ± 110.111.3 × 10−3 ± 1 × 10−454 ± 180.0240.13622.1
Monobactams
AztreonamN.H.N.H.N.H.N.H.N.H.N.H.N.H.N.H.N.H.N.H.N.H.N.H.---
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Joshi, P.; Krco, S.; Davis, S.J.; Asser, L.; Brück, T.; Soo, R.M.; Bodén, M.; Hugenholtz, P.; Wilson, L.A.; Schenk, G.; et al. Phage Anti-Pycsar Proteins Efficiently Degrade β-Lactam Antibiotics. Appl. Biosci. 2024, 3, 438-449. https://doi.org/10.3390/applbiosci3040028

AMA Style

Joshi P, Krco S, Davis SJ, Asser L, Brück T, Soo RM, Bodén M, Hugenholtz P, Wilson LA, Schenk G, et al. Phage Anti-Pycsar Proteins Efficiently Degrade β-Lactam Antibiotics. Applied Biosciences. 2024; 3(4):438-449. https://doi.org/10.3390/applbiosci3040028

Chicago/Turabian Style

Joshi, Pallav, Stefan Krco, Samuel J. Davis, Lachlan Asser, Thomas Brück, Rochelle M. Soo, Mikael Bodén, Philip Hugenholtz, Liam A. Wilson, Gerhard Schenk, and et al. 2024. "Phage Anti-Pycsar Proteins Efficiently Degrade β-Lactam Antibiotics" Applied Biosciences 3, no. 4: 438-449. https://doi.org/10.3390/applbiosci3040028

APA Style

Joshi, P., Krco, S., Davis, S. J., Asser, L., Brück, T., Soo, R. M., Bodén, M., Hugenholtz, P., Wilson, L. A., Schenk, G., & Morris, M. T. (2024). Phage Anti-Pycsar Proteins Efficiently Degrade β-Lactam Antibiotics. Applied Biosciences, 3(4), 438-449. https://doi.org/10.3390/applbiosci3040028

Article Metrics

Back to TopTop