Origin, Diversity, and Multiple Roles of Enzymes with Metallo-β-Lactamase Fold from Different Organisms

β-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B β-lactamases are members of a superfamily of metallo-β-lactamase (MβL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MβL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MβL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as β-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MβL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MβL fold enzymes with demonstrated enzymatic or non-enzymatic activities.


Introduction
Initially discovered in bacteria due to their effectiveness against antibiotics with therapeutic interest in humans, β-lactamases are a group of enzymes capable of degrading several β-lactam antibiotics [1,2]. They are a typical example of the artificial naming of enzymes that have, in reality, multiple potential functions, and this nomenclature has prevented the exploration of their activities and their presence in other organisms or microorganisms. Furthermore, the nomenclature used to describe proteins of the same family may vary depending on the method adopted by researchers, thus enzymes belonging to the same family may be labelled as ribonucleases (RNases), nucleases (DNases), hydrolases, or β-lactamases, depending on the automatic protein annotation, which relies on the initial sequence hits obtained through Blast analysis. The study of the ancestry of β-lactamase motifs has shown that they exhibit some of the oldest enzymatic motifs in the world [3]. Once the antiquity of this type of enzyme has been recognised, the search for sequences which are unrelated to this group of β-lactamases becomes more difficult, due to their early divergence from the ancestral structure. As such, the use of more sensitive approaches

Reported Activities of Bacterial MβL Enzymes other Than β-Lactams Hydrolysis
Besides their hydrolytic activities on β-lactam antibiotics, some bacterial MβL enzymes have been reported with other enzymatic activities as a result of the striking similarity between their protein structures and other enzymes including ribonuclease, nuclease, and lactonase enzymes. Indeed, as reported recently, the classical bacterial MβL IMP-1 enzyme, which hydrolyses all β-lactams including carbapenems, exhibits significant

Reported Activities of Bacterial MβL Enzymes Other Than β-Lactams Hydrolysis
Besides their hydrolytic activities on β-lactam antibiotics, some bacterial MβL enzymes have been reported with other enzymatic activities as a result of the striking similarity between their protein structures and other enzymes including ribonuclease, nuclease, and lactonase enzymes. Indeed, as reported recently, the classical bacterial MβL IMP-1 enzyme, which hydrolyses all β-lactams including carbapenems, exhibits significant protein structure similarity with tRNase Z, a tRNA 3 processing endoribonuclease of the MβL superfamily from Thermotoga maritima. Its enzymatic characterisation demonstrates a significant RNA-hydrolysing activity on both cellular RNA and synthetic small unstructured RNAs [29]. Interestingly, while this study was being published, our research was beginning to reveal the ribonuclease and nuclease ability of the IMP-1 homologous enzyme i.e., class B NDM-1, described in almost all gram-negative bacteria, which significantly hydrolyses in vitro bacterial RNA and single-strand DNA substrates (Supplementary Figure S1). Moreover, while both bacterial MβL enzymes mentioned above can interact with RNA and/or DNA, others such as the ThnS enzyme can exhibit additional activities, such as the hydrolysis of ascorbic acid, as a result of its similarity with UlaG enzymes [30]. Indeed, as we reported recently, while the thnS gene, part of the thienamycin (now chemically modified into imipenem in human medicine) biosynthesis gene cluster from Streptomyces cattleya, is annotated as putative β-lactamase with no reported proof of this activity. We demonstrated its specific hydrolase activity and UlaG high affinity with imipenem in comparison with the other β-lactams (e.g., penicillin G and cefotaxime). As a result of the phylogenetic tree and conserved motif analyses, the ThnS enzyme appears to be a member of the superfamily of MβL fold enzymes, showing additional activities of ribonuclease, nuclease, and hydrolysis of ascorbic acid [30].
Recently, an MβL fold enzyme (BLEG-1) has been reported in the Bacillus lehensis G1 strain, exhibiting significant sequence similarity and activity with the B3 subclass of bacterial MβLs, despite its evolutionary divergence from them [31,32] (Table 1). Upon analysing the phylogenetic tree and comparing the protein structures, it was discovered that the enzyme possessed an active site that was remarkably similar to those found in both the L1 B3 MβL from Stenotrophomonas maltophilia and the glyoxalase II enzymes (YcbL and GloB) from Salmonella enterica. Interestingly, the enzymatic characterisation of the purified BLEG-1 protein demonstrates its dual β-lactams hydrolysis (e.g., ampicillin hydrolysis) and glyoxalase activities [31]. The authors identify an insertion of two amino acids into the active-site loop at the N-terminal region of the BLEG-1 protein and suggested an evolution of the BLEG-1 enzyme from glyoxalase II to the adopted MβL fold activity through this insertion of amino acids [31]. Recently, another atypical enzymatic activity of two MβL fold proteins has also been described from a functional metagenomic study of forest soil [34]. In this study, while the authors performed a function-based screening of libraries generated from the whole metagenomic sequence data of forest soil to identify positive phytase activity in E. coli clones, two clones were positive for this phytase activity. Surprisingly, while phytic acid degradation activity has been restricted to only four protein superfamilies, including histidine phosphatases, tyrosine phosphatases, purple acid phosphatases, and β-propeller phosphatases [47,48], the two obtained proteins (MβLp01 and MβLp02) from this metagenome were annotated and identified as genes encoding for metallo-β-lactamase proteins. Sequence analysis confirmed their membership of the MβL fold superfamily of proteins due to their close protein structure homology with the MβL ZipD from E. coli, a zinc phosphodiesterase with a tRNA-processing endonuclease activity [49]. Based on this discovery, the two proteins were subcloned, expressed, and enzymatically tested. As expected, the enzymatic characterisation revealed for both purified proteins an activity on the majority of tested phosphorylated substrates including phytate. Moreover, both purified enzymes were able to confer to recombinant E. coli strains less sensitivity to β-lactam antibiotics, suggestive of a β-lactamase activity, and qualified by the authors as promiscuous activity [34]. This promiscuous β-lactamase activity was also reported from the discovered and identified subclass B3 MβL protein, PNGM-1 from a conducted functional metagenomic analyses of deep-sea sediments predating the era of antibiotics [35,50]. Indeed, the phylogenetic and protein structure analyses of the PNGM-1 protein revealed its membership of the MβL fold superfamily and its structural similarity with the tRNA Z enzyme, and the activities test confirmed a dual enzymatic β-lactamase and ribonuclease activity of this PNGM-1 protein [35].

Reconstruction of Common Ancestral Sequences and Blast Analyses
In our previous research, to investigate MβL sequences from unsuspected microorganisms such as archaea, which are naturally resistant to all antibiotics including β-lactams because they lack peptidoglycans, we performed the reconstruction of ancestral bacterial β-lactamases, especially class B metallo-β-lactamases based on the maximum likelihood phylogenetic tree analysis using 174 class B MβL variants retrieved from the Arg-annot database [60]. Using an inferred MβL fold ancestor sequence as the query term in a BlastP analysis performed against the archaea database, we were able to identify a huge number of archaeal MβL fold sequences, while BlastP analysis, using contemporary bacterial sequences did not detect these archaeal sequences [6]. All detected MβL fold sequences exhibited the MβL signature (i.e., the conserved "HxHxDH" motif) and are widely distributed in different archaeal groups including Methanomicrobia, Thermococci, Archaeoglobi, Methanococci, Thermoplasmata, Thermoprotei, Methanobacteria, Thaumarchaeota, and Asgardarchaeota ( Figure 1).

The Presence of MβL Fold Enzymes in Subgroups of Archaea: Nanoarchaeota and Asgard Groups
As proof of concept, we once again used a MβL fold ancestor sequence (120 aa in size) from a constructed Maximum Likelihood phylogenetic tree with sequences from bacteria, archaea, humans, and CPRs to confirm the existence of these enzymes in archaearelated groups such as Asgard and Nanoarchaeota. As expected, by conducting a BlastP analysis using the inferred MβL fold ancestor sequence as the query term, we were able to detect the presence of these MβL fold enzymes in these groups of microorganisms (Supplementary Tables S1 and S2). In the Asgard group, MβL fold sequences exhibited protein similarity of between 25% and 46.25%, and alignment of between 48% and 100% with the ancestral MβL fold protein sequence. These were distributed across different Asgard sub-groups such as Helarchaeota, Lokiarchaeota, Heimdallarchaeota, Thorarchaeota, Lokiarchaeum, and Odinarchaeota (Supplementary Table S1). In Nanoarchaeota, our analysis identified homologous sequences with similarity of between 26.82% and 40.32% and length alignment from 56% to 100% with the ancestral MβL fold and were detected in only Nanoarchaeota archaeon (Supplementary Table S2). Interestingly, while the MβL signature can be recognised within these sequences (Figure 2), almost all were annotated by default as MβL fold metallo-hydrolases (Supplementary Tables S1 and S2 and Figure 1).

The Presence of MβL Fold Enzymes in Subgroups of Archaea: Nanoarchaeota and Asgard Groups
As proof of concept, we once again used a MβL fold ancestor sequence (120 aa in size) from a constructed Maximum Likelihood phylogenetic tree with sequences from bacteria, archaea, humans, and CPRs to confirm the existence of these enzymes in archaearelated groups such as Asgard and Nanoarchaeota. As expected, by conducting a BlastP analysis using the inferred MβL fold ancestor sequence as the query term, we were able to detect the presence of these MβL fold enzymes in these groups of microorganisms (Supplementary Tables S1 and S2). In the Asgard group, MβL fold sequences exhibited protein similarity of between 25% and 46.25%, and alignment of between 48% and 100% with the ancestral MβL fold protein sequence. These were distributed across different Asgard subgroups such as Helarchaeota, Lokiarchaeota, Heimdallarchaeota, Thorarchaeota, Lokiarchaeum, and Odinarchaeota (Supplementary Table S1). In Nanoarchaeota, our analysis identified homologous sequences with similarity of between 26.82% and 40.32% and length alignment from 56% to 100% with the ancestral MβL fold and were detected in only Nanoarchaeota archaeon (Supplementary Table S2). Interestingly, while the MβL signature can be recognised within these sequences (Figure 2), almost all were annotated by default as MβL fold metallo-hydrolases (Supplementary Tables S1 and S2 and Figure 1).

Reported Activities of Archaeal MβL Fold Enzymes
Despite the wide presence and distribution of the MβL fold enzymes in the Archaea domain so far, few of these enzymes have been reported in the literature to have proven enzymatic activity such as the that described from the hyperthermophilic archaeon Sulfolobus tokodaii species [38]. Indeed, this S. tokodaii MβL fold enzyme, exhibiting protein structure similarity with the PqsE enzyme from P. aeruginosa involved in the quorum sensing mechanism [61], has been suggested as being involved in as yet undescribed quorum sensing mechanism in archaea using the quinolone antibiotic as a signalling factor [38] ( Table 1). Another archaeal MβL fold enzyme from Haloferax volcanii, exhibiting sequence similarity with a MβL tRNase Z (a tRNA 3′-endonuclease) has been characterised from the transcriptome analysis of generated mutants [37] (Table 1). Despite its similarity with the tRNase Z enzyme, the H. volcanii MβL fold enzyme has not shown tRNA 3′ processing or exonuclease activity, although its activity associated with the membrane transport mechanism has been proven [37] (Table 1). In another archaeal species, Methanosarcina mazei, the reported enzyme was a MβL tRNase Z involved in tRNA maturation (cleavage

Reported Activities of Archaeal MβL Fold Enzymes
Despite the wide presence and distribution of the MβL fold enzymes in the Archaea domain so far, few of these enzymes have been reported in the literature to have proven enzymatic activity such as the that described from the hyperthermophilic archaeon Sulfolobus tokodaii species [38]. Indeed, this S. tokodaii MβL fold enzyme, exhibiting protein structure similarity with the PqsE enzyme from P. aeruginosa involved in the quorum sensing mechanism [61], has been suggested as being involved in as yet undescribed quorum sensing mechanism in archaea using the quinolone antibiotic as a signalling factor [38] ( Table 1). Another archaeal MβL fold enzyme from Haloferax volcanii, exhibiting sequence similarity with a MβL tRNase Z (a tRNA 3 -endonuclease) has been characterised from the transcriptome analysis of generated mutants [37] (Table 1). Despite its similarity with the tRNase Z enzyme, the H. volcanii MβL fold enzyme has not shown tRNA 3 processing or exonuclease activity, although its activity associated with the membrane transport mechanism has been proven [37] (Table 1). In another archaeal species, Methanosarcina mazei, the reported enzyme was a MβL tRNase Z involved in tRNA maturation (cleavage and polyadenylation of the mRNA) [39] (Table 1). From the archaeal species, i.e., Methanocaldococcus jannaschii, three MβL fold enzymes have been reported with distinct enzymatic activities [41]. In the genome of this methanogenic archaeon M. jannaschii, the three genes (mjRNase J1, mjRNase J2, and mjRNase J3), as a result of the exhibited HxHxDH motif, have been recognised as members of the MβL fold superfamily and their characterisation revealed homologous proteins related to ribonuclease Rnase J enzymes, initially discovered in Bacillus subtilis, which are able to exhibit both endo-and 5 →3 exo-ribonucleolytic activities [62]. Interestingly, their enzymatic characterisation demonstrates optimal activity at 60 • C and 5 →3 exonucleolytic activity for purified mjRNase J1 and mjRNase J3 enzymes while mjRNase J2 protein exhibited endonuclease activity (degrade ssDNA substrate) [41]. Apart from these archaeal MβL fold enzymes with reported activity in the literature, the MβL sequences identified by our BlastP analysis using a common ancestor sequence, are annotated either as MβL fold metallo-hydrolases, hydroxyacylglutathione hydrolases (detoxification glyoxalase II enzymes), or L-ascorbate metabolism protein UlaG ( Figure 1).
Interestingly, while all these archaeal enzymes are annotated as "metallo-β-lactamases", none so far have been tested for their hydrolytic activity on β-lactam antibiotics. This was the goal of our recent study, in which the MβL sequence identified from M. barkeri (MetbaB) ( Table 1) was synthesised, cloned into the E. coli BL21(DE3) strain, and expressed in order to evaluate its different putative enzymatic activities including β-lactamase, ribonuclease, nuclease, and glyoxalase [6]. Comparison of the three-dimensional (3D) structure of the expressed protein revealed its high structural similarity with the bacterial New Delhi metallo-β-lactamase (NDM-1) from Klebsiella pneumonaie. As expected, the expressed protein was able to significantly hydrolyse β-lactam substrates including nitrocefin (a chromogenic cephalosporin) and penicillin G. In addition to the β-lactamase activity which was detected, this M. barkeri MβL fold enzyme was also able to significantly hydrolyse bacterial and synthetic RNA substrates, while no nuclease activity was found [6]. As reported in this study, the conducted phylogenetic analysis reveals that the studied archaeal MβL fold protein appeared phylogenetically related to glyoxalase II enzymes. As a result of this relationship, the MetbaB enzyme was tested and weak glyoxalase activity was detected [6].

MβL Fold Enzymes Described in the Eukaryote Domain of Life
In the literature, MβL fold proteins such as hydroxyacylglutathione hydrolase (glyoxalase II), arylsulfatase, DNA and RNA interacting enzymes, nucleotide phosphodiesterases, and CMP-N-acetylneuraminate monooxygenases, have been identified in different eukaryotes including Arabidopsis thaliana, Saccharomyces cerevisiae, Drosophila melanogaster, Bos taurus, Mus musculus, and Homo sapiens [26,63]. In 2016, eighteen MβL fold enzymes had been mentioned in the literature as being present in human cells [44]. These enzymes are polycistronic proteins in which the MβL fold domain can be recognised as a result of their MβL signature, as shown in Figure 2. These 18 human MβLs (hMβLs) are reported in three groups of enzymes: Group 1 relates to the glyoxalase II subfamily and consists of seven enzymes (HAGH, HAGHL, ETHE1, LACTB2, MβLAC1, MβLAC2, and PNKD enzymes) and is constitutively involved in cellular detoxification processes; Group 2 relates to the DNA/RNA interacting subfamily of enzymes composed of nine enzymes (SNM1A, SNM1B, SNM1C, ELAC1, ELAC2, CPSF73, CPSF100, CPSF73L, and INTS9), and Group 3 relates to other hMβLs, in which two enzymes have been reported, namely the NAPE-PLD gene encoding for N-acyl-phosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD), and the CMAH gene, encoding for cytidine monophospho-N-acetylneuraminic acid hydroxylase [44].

Reported Enzymatic Activities of Human MβL Fold Enzymes
Interestingly, in addition to their natural activities in human cells, some of these hMβLs, including the SNM1A and SMN1B enzymes, have been described alongside other enzymatic activities such as the hydrolase activity on anti-cancer drugs like mitomycin C and cisplatin [64,65]. Surprisingly, while the term "metallo-β-lactamase" has been used to annotate these proteins, even for human proteins, their activity against β-lactam antibiotics has thus far not been reported.
In the same way, as we did for archaea, we reported the evaluation of enzymatic activities of four selected hMβL proteins, as a result of their conserved MβL "HxHxDH" motif and histidine residues (H196 and H263), as seen in bacterial MβLs (Table 1) [43]. These four hMβL enzymes were the MBLAC2 protein associated with the biosynthesis of Bcell exosomes [42], the endoribonuclease LACTB2 protein [66], SNM1A, and SNM1B, with a function of DNA cross-link repair enzymes (nucleases) [67]. In this study, the proteins which were synthetised and optimised for expression in E. coli BL21(DE3) strains were tested against β-lactam antibiotics and, as expected, while, no activity was detected for the LACTB2 protein, the MBLAC2, SNM1A, and SNM1B proteins were able to significantly hydrolyse nitrocefin and penicillin G, and this activity was inhibited by a β-lactamase inhibitor (sulbactam) [43].

Discovery of MβL Fold Enzymes in Giant Viruses
With the existence of evidence of MβL fold enzymes in all domains of life, including Bacteria, Archaea, and Eukaryotes, as mentioned above, we similarly conducted, as part of our research into these MβL fold enzymes, a search for MβL fold enzymes in Giant viruses. By searching for the conserved MβL motif against a protein database of giant viruses retrieved from the NCBI (n = 72,993 proteins), we were able to identify fifteen confirmed MβL fold sequences (based on a protein length of at least 200 aa). Default protein annotations of these gvMβL sequences from the NCBI database included ribonuclease Z, the ribonuclease BN/tRNA processing enzyme, the MβL fold metallo-hydrolase/oxidoreductase superfamily, the metallo-β-lactamase superfamily protein, the Ankyrin repeat domain-containing protein, and proteins with unknown function. However, as shown in Figure 1, while most of these gvMβL sequences grouped together within the phylogenetic tree, interestingly three gvMβLs branched within bacterial MβLs, suggestive of a horizontal exchange between bacteria and giant viruses, especially Pandoravirus dulcis and Pandoravirus salinus. Two other gvMβLs appear to be clustered with sequences from humans and one gvMβL grouped with Asgard MβL fold sequences (Figure 1).

Description of Dual Enzymatic Activity of a Giant Virus MβL Fold Enzyme
To the best of our knowledge, no studies have yet reported the activities of these MβL fold enzymes from a giant virus. Thus, in our recent work, we characterised the enzymatic activities of a MβL fold protein from giant Tupanvirus deep ocean (TupBlac protein), naturally involved in translation mechanisms in giant viruses. Protein analysis using a conserved domain search (CD Search) tool [68] revealed its membership of the ribonuclease Z group (the tRNA-processing endonuclease enzymes) and conserved motif analysis revealed its MβL signature (HxHxDH motif) and conserved residues (H60-H62-H65) [45]. As described in that study, the expressed TupBlac protein from the E. coli BL21(DE3) strain and its enzymatic characterisation demonstrated a dual β-lactamase and ribonuclease activity, since the TupBlac protein was able to hydrolyse nitrocefin, penicillin G, and RNA substrate (of bacteria or Acanthamoeba castellanii) [45] (Table 1).

Wide Diversity of MβL Fold Enzymes in the CPR Domain
As described in 2015, as a result of the power of deep sequencing methods and bioinformatic analyses, a new branch of microorganisms from the tree of life was discovered and named Candidate Phyla Radiation (CPR) [69,70]. This group of microorganisms, in addition to their symbiotic lifestyle with bacteria, is characterised by their small size (100 to 300 nm), reduced genomes (≈1-Mb), the lack of biosynthetic pathways, and their abundance in all environments and various human microbiomes [46,69]. As recently reported, we investigated the existence of the MβL fold enzymes in this new domain of life as part of the investigation into the resistomes of CPR microorganisms. Indeed, the in silico analyses, based on BlastP and functional domain prediction of 4062 CPR genomes to look for the presence of antibiotic resistance (AR)-like enzymes revealed highly equipped microorganisms with more than 85 different AR-like enzymes against 14 different classes of antimicrobials including aminoglycosides, glycopeptides, and β-lactams [71]. Among these AR-like enzymes, especially β-lactam resistance proteins, the search for an MβL signature (HxHxDH) revealed their existence in this domain of life, as shown in Figure 2 for representative sequences. The default annotations of these CPR proteins from the NCBI database are mainly "MβL fold metallo-hydrolase", "β-lactamase domain protein", or "RNA-metabolising metallo-β-lactamase". Interestingly, as shown in Figure 1, the identified MβL fold sequences in CPR microorganisms constitute one branch from the phylogenetic tree in which archaeal and Asgard sequences can be identified, and one of them, from Sulfolobus tokodaii, was reported to have a function associated with the quorum sensing mechanism [38]. This may suggest a native function of these CPR MβL fold enzymes, associated with communication processes with bacteria, with which they share an obligate symbiotic lifestyle, as reported in the literature [70].

Reported Enzymatic Activity of CPR MβL Fold Enzymes on β-Lactam Antibiotics
Despite the default annotation of these enzymes, which may suggest a hydrolytic activity on antibiotics or RNA/DNA substrates, no studies have thus far reported enzymatic characterisation of these proteins. Thus, after we reported the presence and wide distribution of the MβL fold sequences in CPR microorganisms, we experimentally expressed, purified, and tested the enzymatic activity of five selected β-lactamase proteins, including class A and class B metallo-β-lactamases from CPR, to evaluate their ability to hydrolyse various substrates including β-lactams, RNA, and DNA [46] ( Table 1). The threedimensional (3D) structural analysis of these five CPR β-lactamases confirms (with 100% confidence) their structural similarity with bacterial class A β-lactamase, metal-dependent hydrolases of the β-lactamase superfamily II, human metallo-β-lactamase containing protein 1, ribonuclease J1, and dual endo-and exonuclease enzymes [46]. This study is the first and only piece of research that has reported on the β-lactam and RNA hydrolysing activity of MβL fold proteins from CPR groups.

Discussion
In this review, we have highlighted the considerable extension of core genes for βlactamases into all domains of life, as shown in Figure 3A. Metallo-β-lactamase fold proteins appear to be "multifunctional enzymes" with hydrolytic, non-hydrolytic, or non-enzymatic activities, as the literature reports at least thirteen demonstrated and distinct activities (Figure 4), and it is difficult to know what the initial activity was. It can be speculated that the primary activity was that of nuclease/ribonuclease, the first role of which was to digest unused or parasitic DNA/RNA in cellular metabolism as reported in bacteria, in humans, and in archaea [41,72,73]. The potential of these hydrolases was later used for different functions. β-lactamases are one of the most interesting examples in the history of science, as they were first discovered for their activity on β-lactam antibiotics, explaining bacterial resistance against these drugs [1,2]. It is plausible that the nomenclature of these enzymes has prevented the exploration of their real functions in organisms/microorganisms other than bacteria. β-lactamase fold sequences were recently identified for the first time in the human genome, while their default functions have been associated with various biological processes [44,66]. Indeed, the identification of genes encoding for β-lactamases in human genomes has not been explored for long, which has prevented the existence of β-lactamase activity in human cells that could inactivate penicillin G in these cells [43]. A great deal of confusion can be seen in the naming of these MβL fold enzymes as, according to the first Blast hit during the initial protein annotation, these latter are considered to have a unique and essential enzymatic activity instead taking into account other potential activities of these enzymes. For example, we initially had great difficulty in publishing the metallo-βlactamase fold enzyme from archaea (from Methanosarcina barkeri) [6], given that it made no sense for archaea microorganisms to host β-lactamase enzymes, which were naturally resistant to β-lactams, based on the lack of β-lactam targets in their cell wall [74,75]. We can speculate that the existence or role of these MβL fold enzymes in archaea may be associated with other biological functions such as the RNA/DNA metabolism or the use of β-lactam antibiotics after enzymatic degradation as a source of carbon for nutrients, as described in some bacteria such as Pseudomonas, Burkholderia, and Pandoreae [76,77].
the first time in the human genome, while their default functions have been associated with various biological processes [44,66]. Indeed, the identification of genes encoding for β-lactamases in human genomes has not been explored for long, which has prevented the existence of β-lactamase activity in human cells that could inactivate penicillin G in these cells [43]. A great deal of confusion can be seen in the naming of these MβL fold enzymes as, according to the first Blast hit during the initial protein annotation, these latter are considered to have a unique and essential enzymatic activity instead taking into account other potential activities of these enzymes. For example, we initially had great difficulty in publishing the metallo-β-lactamase fold enzyme from archaea (from Methanosarcina barkeri) [6], given that it made no sense for archaea microorganisms to host β-lactamase enzymes, which were naturally resistant to β-lactams, based on the lack of β-lactam targets in their cell wall [74,75]. We can speculate that the existence or role of these MβL fold enzymes in archaea may be associated with other biological functions such as the RNA/DNA metabolism or the use of β-lactam antibiotics after enzymatic degradation as a source of carbon for nutrients, as described in some bacteria such as Pseudomonas, Burkholderia, and Pandoreae [76,77].   Our recent work highlights a wide distribution and great conservation of the MβL fold proteins in archaea [6]. By using a more sensitive search approach such as the use of common ancestor sequences in blast analysis, we can identify these MβL fold proteins in archaea-related microorganisms such as Nanoarchaeota and Asgard, as highlighted in this review (Figure 1). This approach demonstrates and confirms the power of using a common ancestor to identify distant homologous sequences hosted by unsuspected microorganisms as demonstrated for giant viruses for which the distance between the reconstructed RNA polymerase (RNAP) ancestor and RNAPs of bacteria, archaea, eukarya, and megavirales were significantly shorter than the distance between RNAPs from each organism [84]. Indeed, as can be seen in Figure 3B, the pairwise comparison performed on the similarity between MβL sequences from the different domains of life with the inferred common MβL fold ancestor sequence demonstrated a higher percentage of similarity Our recent work highlights a wide distribution and great conservation of the MβL fold proteins in archaea [6]. By using a more sensitive search approach such as the use of common ancestor sequences in blast analysis, we can identify these MβL fold proteins in archaea-related microorganisms such as Nanoarchaeota and Asgard, as highlighted in this review ( Figure 1). This approach demonstrates and confirms the power of using a common ancestor to identify distant homologous sequences hosted by unsuspected microorganisms as demonstrated for giant viruses for which the distance between the reconstructed RNA polymerase (RNAP) ancestor and RNAPs of bacteria, archaea, eukarya, and megavirales were significantly shorter than the distance between RNAPs from each organism [84]. Indeed, as can be seen in Figure 3B, the pairwise comparison performed on the similarity between MβL sequences from the different domains of life with the inferred common MβL fold ancestor sequence demonstrated a higher percentage of similarity between the ancestor MβL and sequences from the different organisms in comparison with any pairs of sequences from two different organisms.

Conclusions
The evidence of several activities including β-lactamase, nuclease, ribonuclease, lactonase, glyoxalase, phytase, and potentially other unidentified hydrolase activities from enzymes of the same family highlights the significance of accurately naming enzymes in order to understand their nuclear reactivity. This is particularly challenging due to the scarcity of competent reviewers outside the bacterial world able to evaluate knowledge extension beyond the initial enzyme activity identification, which remains extremely problematic. Classical Blast analyses present some limitations, for example, when it comes to identifying remote homologous sequences, because of the few similarities between them. This is why alternative search tools are now being proposed by NCBI to improve the shortcomings of the classical BlastP (i.e., PSI-BLAST: Position-Specific Iterated BLAST; PHI-BLAST: Pattern Hit Initiated BLAST; DELTA-BLAST: Domain Enhanced Lookup Time Accelerated BLAST). More sensitive search approaches including the search for Hidden Markov Models (HMM) profile, Sequence Similarity Network analysis (SSN), and the use of inferred common ancestor sequences as targets are more appropriate and more useful when it comes to detecting and identifying homologous sequences in any organism. This review highlights a great example of the misleading annotations of proteins in public sequence databases, which is not only limited to MβL fold proteins but applied to many classes of sequences proteins as previously reported, greatly reducing our perception of the multifunctionality of some proteins, due to the unique attributed function by the default protein annotation [85]. In essence, this history of β-lactamases has epistemological importance which we believe is essential.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12131752/s1, Figure S1: Ribonuclease and nuclease activities of the New Delhi Metallo-β-lactamase 1 (NDM-1). (A) bacterial RNA substrate hydrolysed by the NDM-1 enzyme in the presence and absence of inhibitor (i.e., sulbactam and EDTA). (B) Single strand bacterial DNA (ssDNA forward and reverse) hydrolysed by the NDM-1 enzyme while double strand DNA (dsDNA) was not hydrolysed. H 2 O, Blank, and GO (Glycine oxidase) have been used as negative controls, and bacterial DNase has been used as positive control. The methodology and material used for this experiment have been reported in our previous work [30]. Table S1: BlastP result of the MβL fold ancestor (120 aa) sequence against the Asgard group database (NCBI) (94 hits); Table S2: BlastP results of the MβL fold ancestor (120 aa) against the Nanoarchaeota database (NCBI) (40 hits).