Dual activity of PNGM-1, a metallo-β-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-β-lactamases

Antibiotic resistance is a steadily increasing global problem which could lead to a fundamental upheaval in clinical care with the potential to return us to the pre-antibiotic era1-4. The production of β-lactamases, a group of enzymes that confer antibiotic resistance in Gram-negative bacteria, is now one of the major barriers in treating Gram-negative infections5. β-Lactamases are classified according to their catalytic mechanisms into serine β-lactamases and metallo-β-lactamases6,7. There are functional and structural similarities between serine β-lactamases and penicillin-binding proteins, and so serine β-lactamases are thought to have evolved from a penicillin-binding protein7,8. Given the functional and structural differences between serine β-lactamases and metallo-β-lactamases, metallo-β-lactamases are thought to have evolved from a protein other than a penicillin-binding protein, but to date this ancestor remains unknown8-11. We discovered PNGM-1, the first subclass B3 metallo-β-lactamase, in deep-sea sediments that predate the antibiotic era12. Here we discover the dual activity of PNGM-1, pinpointing the evolutionary origin of subclass B3 metallo-β-lactamases. Phylogenetic analysis suggested that PNGM-1 could yield insights into the evolutionary origin of subclass B3 metallo-β-lactamases. We reveal the structural similarities between tRNase Zs and PNGM-1, which prompted us to investigate their evolutionary relationship and the possibility of them possessing dual enzymatic activities. We demonstrate that PNGM-1 has dual activity with both true metallo-β-lactamase and tRNase Z activity, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 metallo-β-lactamases and tRNase Zs. These comparisons revealed that the B3 metallo-β-lactamase activity of PNGM-1 is a promiscuous activity and subclass B3 metallo-β-lactamases are thought to have evolved through PNGM-1 activity. Our work provides a foundation for the evolution of tRNase Z into subclass B3 metallo-β-lactamases through the dual activity of PNGM-1.


Introduction
β-Lactam antibiotics are one of the most successful drugs used for the treatment of bacterial infections and represent roughly 65% of the total world market for antibiotics 13 . Therefore, resistance to β-lactam antibiotics is one of the most serious problems associated with Gram-negative bacterial infections 5 . β-Lactamases are produced by various bacteria conferring them resistant to β-lactam antibiotics such as penicillins, cephalosporins, monobactams, and/or carbapenems (the last resort drugs for treating bacterial infections) 9,14 . β-Lactamases are divided into four classes, A-D. The enzymes belonging to classes A, C, and D, are serine β-lactamases, which are thought to originate from a penicillin-binding protein 7,8 . Class B enzymes, which are further divided into three subclasses, B1-B3 6 , are metallo-β-lactamases (MBLs) that hydrolyse almost all β-lactam antibiotics, including carbapenems, thereby representing a critical antibiotic resistant threat 10,11 . The evolutionary origin of MBLs is currently unknown.
Subclass B1 and B3 MBLs require two zinc ions for maximum β-lactamase activity, whereas the subclass B2 MBLs require only one 15 . Recently, we discovered a novel B3 MBL named PNGM-1 (Papua New Guinea Metallo-β-lactamase), which was the first B3 enzyme obtained from a functional and bacterial metagenomic library of deep-sea sediments from the Edison seamount, which existed prior to the antibiotic era 12,16 .
A member of the MBL superfamily, tRNase Z, is a tRNA processing enzyme which removes the 3′ trailer from pre-tRNA 24,25 . Most tRNase Zs cleave pre-tRNA immediately downstream of a discriminator nucleotide (nt), onto which the CCA residues are added to produce mature tRNA. tRNase Zs are categorized into two groups, tRNase Z S containing 300-400 amino acids and tRNase Z L containing 800-900 amino acids. Bacteria and archaea have tRNase Z S only, while eukaryotes have either tRNase Z L only or both tRNase Z S and tRNase Z L . tRNase Z (prokaryotic tRNase Z S ) can cleave unstructured single-strand RNAs that are unrelated to pre-tRNA in vitro 26 .
The tRNase Z catalytic center is formed by the major five well conserved residues (His-48, His-50, Asp-52, His-53 and His-222 in the case of T. maritima tRNase Z) together with two zinc ions. Residues Asp-52 and His-222 are thought to directly contribute as donors during the catalytic proton transfer 27 .
In this paper, we found that in addition to β-lactamase activity, PNGM-1 possesses endoribonuclease (tRNase Z) activity on both pre-tRNA substrates and on small unstructured single-strand RNA substrates. Our functional, phylogenetic and structural analyses of PNGM-1 and their comparison with the properties of other MBLs (proteins containing the αββα-fold with true-β-lactamase activity) and structurally representative MBL fold proteins (proteins having αββα-fold without true-β-lactamase activity) of the MBL superfamily, reveal the evolution of tRNase Z to subclass B3 MBLs through the dual activity of PNGM-1.

Strains and plasmids
Escherichia coli BL21 (DE3) and plasmid-containing E. coli strains were used for all cloning and expression studies. The pET-28a(+)/His6-PNGM-1 plasmid has been described previously 12 . The strains and plasmids used in this study are listed in Supplementary Table S1.

Construction of PNGM-1 mutants by site-directed mutagenesis
Site-directed mutagenesis was carried out using a QuikChange II  amino acids) were prepared as previously described 12,24,28 .

Steady-state kinetic analysis
Kinetic assays were conducted at 30°C with a Shimadzu UV-1650PC  Table S3.

Structure determination of PNGM-1
Crystallization and X-ray diffraction data collection of PNGM-1 was carried out as previously published 16  X-ray diffraction data were collected to 2.1 and 2.3 Å resolutions for the native and SeMet PNGM-1 crystals, respectively. All data were integrated and scaled using the DENZO and SCALEPACK crystallographic data-reduction routines 31 . The PNGM-1 structure was solved by the SAD method using SeMet PNGM-1. The interpretable electron density was obtained at 2.3 Å resolution for the SeMet PNGM-1 data set using the single wavelength SAD protocol of AUTO-RICKSHAW, an automated crystal structure determination platform 32 in the P21 space group. The native PNGM-1 structure was determined by molecular replacement with MOLREP 33 using the SeMet PNGM-1 structure (Supplementary Table S4).

In vitro RNA cleavage assay
To investigate the β-lactam-hydrolysing activity of the four PNGM-1 mutants against β-lactam antibiotics, the catalytic properties of purified PNGM-1 and the four PNGM-1 mutants were assessed (Table 1). Kinetic assays revealed that all four mutants were unable to hydrolyze β-lactam antibiotics including extended-spectrum cephalosporins (ceftazidime and cefotaxime) and carbapenems (meropenem, imipenem and ertapenem). Substitution of residues in the metal binding motif had the most significant influence on the catalytic properties of PNGM-1 against cephalosporins and carbapenems. Therefore, this result indicated that PNGM-1 possesses true β-lactamase (MBL with its ability to hydrolyse carbapenems) activity.

The insight into the evolutionary origin of subclass B3 MBLs
The evolutionary origin of subclass B3 MBLs has not yet been identified.

Structural similarity between PNGM-1 and tRNase Z
To examine the evolutionary relationship between PNGM-1 and tRNase Z, we compared the PNGM-1 structure with those of structurally representative tRNase Zs from B. subtilis, E. coli and T. maritima (Fig. 2). All the structures have the characteristic αββα-fold of the MBL superfamily with the unique metal binding motif. . tRNase Z has a conserved tRNA-binding arm, which is lost in PNGM-1 and subclass B3 MBLs (Fig. 2). These structural similarities between tRNase Zs and PNGM-1 prompts us to investigate the possibility of PNGM-1 possessing tRNase Z activity.

PNGM-1 possesses RNase activity
To test whether PNGM-1 possesses RNase activity, we examined whether accumulated under both conditions (Fig. 4). The RNA integrity numbers in the assays with Mg 2+ and Mn 2+ decreased by 25% and 18%, respectively. These results suggest that PNGM-1 possesses a ribonuclease activity.

PNGM-1 can cleave unstructured RNAs endoribonucleolytically
Next, we examined PNGM-1 for RNase activity on two small RNA substrates.
The unstructured RNAs, usRNA1 (24 nt) and usRNA9 (22 nt), were used as they are cleaved by tRNase Z. An unstructured 24-nt DNA (usDNA1), corresponding to usRNA1 was used as a negative control substrate. These three substrates, which were 5′-labeled with 6-carboxyfluorescein, were incubated with 15 μM PNGM-1 in the presence of 10 mM Mg 2+ at 50 ˚C for 30-90 min, and the products were analysed by denaturing polyacrylamide gel electrophoresis. Fragments of various sizes were generated from usRNA1 and usRNA9 but not from usDNA1 (Fig. 5a). The pattern of generated RNA fragments suggest that PNGM-1 possesses an endoribonuclease activity, not an exoribonuclease activity, and has no DNase activity.
The amounts of usRNA1 and usRNA9 cleavage products increased in a dose-dependent manner, with the optimal temperature for RNase activity estimated to be around 50 ˚C (Fig. 5b, c). RNase activity was also assessed by varying the concentration (5-50 mM) of MgCl2 or MnCl2, and the optimal concentrations of Mg 2+ and Mn 2+ were estimated to be around 10 mM and 5-10 mM, respectively (Fig. 5d).

Asp-95 and His-257 are essential for the RNase activity of PNGM-1
To rule out the possibility that the observed RNase activity of PNGM-1 originates from unidentified contaminant RNases, we examined the five PNGM-1 mutants. These mutants contained single amino-acid substitutions of residues that were likely essential for RNase activity on usRNA1 and usRNA9. The PNGM-1 mutants (H91A, H93A, D95A, H96A and H257A: five mutants with alanine replacements of His-48, His-50, Asp-52, His-53 and His-222 at structurally equivalent positions in Tm-tRNase Z, respectively) had a single substitution of alanine for histidine or aspartic acid. The corresponding amino-acid substitutions in Tm-tRNase Z are known to abolish its activity without Mn 2+ ions. All five PNGM-1 mutants showed little or no RNase activity in the presence of Mg 2+ (Fig. 6a). These results indicate that the observed RNase activity of PNGM-1 is genuine.
RNase activity was recovered, albeit inefficiently, in the presence of Mn 2+ for mutants H91A, H93A and H96A but not D95A and H257A (Fig. 6b). This Mn 2+ -rescue phenomenon, which was first observed for the pre-tRNA cleavage reaction by

Tm-tRNase Z, suggests that Asp-95 (Asp-52 in Tm-tRNase Z) and His-257 (His-222 in
Tm-tRNase Z) are essential and directly contribute to proton transfer for the RNase activity of PNGM-1, further corroborating the RNase activity of PNGM-1. Interestingly, the catalytic efficiency of H257A for all tested β-lactams decreased in comparison to that of wild-type PNGM-1 (Table 1), which suggests that H257 (as well as H91A, H93A, D95A and H96A) of PNGM-1 plays an important role in both its RNase and β-lactamase activities.

Cleavage site preference of the endoribonucleolytic activity of PNGM-1
We examined the cleavage site preference of the endoribonucleolytic activity of PNGM-1 on usRNA1 and usRNA9. The cleavage reaction products for usRNA1 and usRNA9 were analysed at nt resolutions, with corresponding substrate ladders, on a 20% polyacrylamide denaturing gel. The cleavage patterns of usRNA1 and usRNA9 by PNGM-1 were unique and different from those for Tm-tRNase Z (Fig. 7). PNGM-1 appears to have a tendency to cleave the RNA substrates between pyrimidine nucleotides. The slightly different cleavage patterns observed for Tm-tRNase Z compared with those in the previous study 26 could be due to the different assay conditions.

PNGM-1 can process a pre-tRNA substrate
To test whether PNGM-1 can process pre-tRNA similar to tRNase Z, the 84-nt human pre-tRNA Arg , which was 5′-labeled with fluorescein, was used as a pre-tRNA substrate (Fig. 8a). The pre-tRNA Arg was incubated with PNGM-1, Tm-tRNase Z or human Δ30 tRNase Z L , with cleavage products analysed by denaturing polyacrylamide gel electrophoresis. PNGM-1 cleaved the pre-tRNA Arg similar to Tm-tRNase Z and human Δ30 tRNase Z L (Fig. 8b). Major cleavage by PNGM-1 occurred after the 76th nt (3 nt downstream of the discriminator), whereas major cleavage by Tm-tRNase Z and human Δ30 tRNase Z L occurred after the 75th nt and the discriminator (73rd nt), respectively, as expected 24,25,27 . Therefore, PNGM-1 is the first unique enzyme that has dual activity i.e., true β-lactamase (MBL hydrolysing all tested and clinically-used β-lactam antibiotics; Table 1) and tRNase Z activity. We are currently trying to co-crystallise PNGM-1 or the PNGM-1 mutants with carbapenems or tRNA Arg in an attempt to understand how PNGM-1 possesses this dual activity.

Origin of subclass B3 MBLs
Our functional analyses of PNGM-1 showed that PNGM-1 has true β-lactamase (MBL) and tRNase Z activity (Table 1 and Figs. 3-7). We demonstrate the dual activity of PNGM-1, which strongly suggests that PNGM-1 has evolved from a tRNase Z [MBL fold protein with a single native activity and without true-β-lactamase activity (Supplementary Table S5)]. This is in agreement with earlier reports suggesting that novel enzymes could evolve from an existing enzyme with single native activity through the recruitment of multiple functions (e.g., dual activity) 20 Table S6). Analysis of the superimposed structures of these six enzymes with PNGM-1 showed the gradual change from tRNase Z to subclass B3 MBL (Fig. 2). tRNA is a bulky substrate similar to tRNase Z enzyme monomer. In order to recognize tRNA, tRNase Z has an elongated dimeric structure with an extended tRNA-binding arm protruding from the central β-sheet in the αββα-fold (Fig. 2). In the dimer structure, subunit A is responsible for substrate recognition while subunit B is catalytically active (Fig. 2). The tRNA-binding arm has a positively charged surface which provides an extra binding affinity for the negatively charged tRNA phosphate backbone ( Supplementary Fig. S1). PNGM-1 has maintained the dimerizing ability but has lost the tRNA-binding arm. Subclass B3 MBLs has lost the dimerization. However, the dimerization of subclass B3 MBLs is not necessary for the binding of much smaller β-lactam substrates. When tRNA was modelled to bind to subclass B3 MBLs, subclass B3 MBLs are unable to interact with tRNA except the 3' end of tRNA to be processed ( Fig. 2). Therefore, among subclass B3 MBLs, only PNGM-1 has true tRNase Z activity.
We compared the PNGM-1 structure with carbapenem-bound subclass B3 MBL structures such as diapenem-bound CphA and doripenem-bound SMB-1 ( Supplementary Fig. S2)   17,20,42 . These evolutionary processes help us to understand where MBL genes came from and predict the future evolution of MBL genes, as previously described 40 . Kinetic data for PNGM-1 were identical to data from reference 12. b His-257 of PNGM-1 is located at the position corresponding to His-222 of T. maritima tRNase Z. c NH, not hydrolysed. Data are mean ± s.d. of three assays. were repeated at least three times and were reproducible.       were repeated at least three times and were reproducible.