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][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 (compared with classical Blast analysis) to search for distant homologous sequence, such as the reconstruction of a common ancestor sequence, as previously reported for class A β-lactamases ^[4][5][4,5] and using it as a query makes it possible to identify sequences that are too far away from contemporary sequences to be identified in a functional way.

2. Diversity of the Superfamily of Metallo-β-Lactamase (MβL) Fold Enzymes

Within the classification of bacterial β-lactamases, which consist of four classes: A, B, C, and D ^[6][7][7,8], MβL enzymes alone occupy class B which is subdivided into three subclasses (subclass B1/B2/B3) as the result of divergent evolution events. They differ from the other classes by their active site which requires bivalent metal ions (such as Zn²⁺, Fe^2+, Mg²⁺, or Ca²⁺) for their activity ^{[6][8][9][10]}[7,9,10,11]. This catalytic site, characterised by a highly conserved motif (HxHxDH) and residues (H196 and H263), appears to be ancestrally shared by the superfamily of MβL fold proteins (with 34,000 proteins identified to date) with diverse biological functions including β-lactamases, nucleases, ribonucleases, lactonases, glyoxalases, hydrolases, phosphodiesterases, Aryl sulfatases, Alkylsulfatases, CMP-NeuAc hydroxylases, flavoproteins, and others which are distributed across the different domains of life including bacteria, archaea, and eukaryotes ^[11][12][13][12,13,14]. Moreover, as reported, the multifunctionality of these MβL fold enzymes and their affinities to different substrates are supported by the protein variable region of the enzymes (rather than the conserved motif (i.e., HxHxDH) and cofactor dependencies, which are responsible for the modulation of enzymatic activity, specificity, and oligomerisation proteins ^[9][14][10,15]. However, the protein similarity between enzymes of this MβL fold superfamily can be less than 20% ^[12][15][13,16] making it very unlikely that homologous sequences of contemporary bacterial MβLs in the other microorganisms such as Archaea, Giant viruses, Asgard, Nanoarchaeota, or Candidate Phyla Radiation (CPR) will be found. As reported in the “Darwinian Grandparenting” theory, we are genetically closer to our grandparents than to our cousins ^[16][17]; thus, the use of inferred common ancestor sequences as queries to identify homologous bacterial MβLs from not-yet investigated microorganisms represents one of the more pertinent approaches to identifying these enzymes in any domain of life. More sensitive approaches, including reconstruction of the common ancestor sequence ^[5][17][5,18], searching for Hidden Markov Models (HMM) profiles ^[17][18][18,19], Sequence Similarity Network analysis (SSN) ^[12][19][13,20], and 3D structure similarity analysis, have been used to look for homologous MβL enzymes in remote sources. As expected, these approaches appear significantly more flexible than classic Blast analysis in terms of identifying homologous MβL enzymes in unsuspected organisms and/or microorganisms.

3. MβL Fold Enzymes in Bacteria: Class B β-Lactamases

3.1. Distribution and Diversity of MβL Fold Enzymes in Bacteria

In bacteria, these MβL enzymes include more than 325 variants grouped into 63 MβL types and divided into three sub-groups: subgroup B1 (e.g., NDM-1, VIM-2, and IMP-1); sub-group B2 (e.g., CphA1, CphA7, and ImiS); sub-group B3 (e.g., GOB-13, LRA-1, and CAR-1) (Figure 1), based on their differences in amino acid sequences and their catalytic sites which interact with either one or two Zn²⁺ ions ^[20][21][22][21,22,23]. The metallo-β-lactamase enzyme was identified for the first time in 1966, in a non-pathogenic strain of Bacillus cereus with a cephalosporinase activity which was inhibited when pre-incubated with ethylene-diamine-tetra-acetic acid (EDTA) ^[23][24]. Nowadays, MβL enzymes can be identified in more than 50 different bacterial species including gram-negative bacteria such as Enterobacteriaceae spp. and non-fermentative bacteria (such as, Acinetobacter spp. and Pseudomonas spp.), Elizabethkingia spp., Stenotrophomonas spp., Neisseria spp., Aeromonas spp., Shewanella spp., Myroides spp., Pedobacter spp., Empedobacter spp., Bacteroides spp., Vibrio spp., Bradyrhizobium spp., Caulobacter spp., Eristalis spp., Sphingomonas spp., Massilia spp., Burkholderia spp., Pectobacterium spp., and Gemmatimona spp.) ^[20][21][22][21,22,23]. Interestingly, as shown in Figure 1, while all the bacterial B1 and B2 MβL enzymes group together separately to the other domains of life, the GOB type enzymes (sub-group B3), are strongly related with enzymes from archaea and archaea-related microorganisms (i.e., Asgard), suggestive of their origin from this domain of life, and a horizontal transfer has occurred from archaea to a single bacterial group, namely, the Flavobacteriacaea family, especially in Elizabethkingia species. All the bacterial MβLs presented in this phylogenetic tree have been reported with proof of hydrolase activity on β-lactams, especially on carbapenems, and are all described in gram-negative bacteria except one MβL type i.e., BcII MβL enzyme (with seven variants), reported in a single bacterial gram-positive species, Bacillus cereus ^[20][24][21,25]. However, based on the MβL signature, these proteins have also been identified in other Gram-positive bacteria such as Streptococcus pneumoniae, in which MβL fold proteins are recognised as choline-binding proteins, DNA uptake-related proteins (nucleases) and L-ascorbate 6-phosphate lactonase ^[25][26][27][26,27,28]. Interestingly, it has been reported that the enzymatic characterisation of the L-ascorbate 6-phosphate lactonase enzyme from S. pneumoniae ATCC 49136 shows a β-lactamase activity since the purified enzyme is able to hydrolyse both nitrocefin and ampicillin-based antibiotics ^[26][27].

3.2. 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 ^[28][29]. Interestingly, while this study was being published, the ouresearchers' 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 ^[29][30]. Indeed, as thwe researchers 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. ThWe researchers 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 ^[29][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 ^[30][31][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 ^[30][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 ^[30][31]. Recently, another atypical enzymatic activity of two MβL fold proteins has also been described from a functional metagenomic study of forest soil ^[33][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][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 ^[33][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 ^[34][50][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 ^[34][35]. Another example of bacterial MβL fold enzymes with activities other than β-lactam hydrolase is that of lactonase enzymes. Recognised as members of the metallo-β-lactamase superfamily as a result of their conserved HxHxDH MβL motif ^[51][52][51,52], these enzymes are involved in the bacterial quorum sensing mechanism, especially by disrupting bacterial signalling via the enzymatic degradation of acyl homoserine lactone (AHL) molecules ^[13][52][14,52]. The quorum sensing mechanism is described as a communication system used by bacteria to manage large panels of biological processes often related to pathogenicity, such as the production of proteases, or antimicrobial compounds, such as violacein [53]. These enzymes have been reported in various and atypical bacterial species such as thermoacidophilic bacteria including Alicyclobacter acidoterrestris, Geobacillus caldoxylosilyticus, Chromobacterium spp., Bacillus thuringiensis, Escherichia coli, Ruegeria mobilis, Microbacterium testaceum, Muricauda olearia, Arthrobacter sp. and Chryseobacterium spp. ^{[27][51][52][53][54][55][56][57][58][59]}[28,51,52,53,54,55,56,57,58,59].