Structural Study of Metal Binding and Coordination in Ancient Metallo-β-Lactamase PNGM-1 Variants

The increasing incidence of community- and hospital-acquired infections with multidrug-resistant (MDR) bacteria poses a critical threat to public health and the healthcare system. Although β-lactam antibiotics are effective against most bacterial infections, some bacteria are resistant to β-lactam antibiotics by producing β-lactamases. Among β-lactamases, metallo-β-lactamases (MBLs) are especially worrisome as only a few inhibitors have been developed against them. In MBLs, the metal ions play an important role as they coordinate a catalytic water molecule that hydrolyzes β-lactam rings. We determined the crystal structures of different variants of PNGM-1, an ancient MBL with additional tRNase Z activity. The variants were generated by site-directed mutagenesis targeting metal-coordinating residues. In PNGM-1, both zinc ions are coordinated by six coordination partners in an octahedral geometry, and the zinc-centered octahedrons share a common face. Structures of the PNGM-1 variants confirm that the substitution of a metal-coordinating residue causes the loss of metal binding and β-lactamase activity. Compared with PNGM-1, subclass B3 MBLs lack one metal-coordinating residue, leading to a shift in the metal-coordination geometry from an octahedral to tetrahedral geometry. Our results imply that a subtle change in the metal-binding site of MBLs can markedly change their metal-coordination geometry and catalytic activity.

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.

Partitioning of the Nuclear and Mitochondrial tRNA 3′-End Processing Activities between Two different Proteins in Schizosaccharomyces pombe

tRNase Z is an essential endonuclease responsible for tRNA 3′-end maturation. tRNase Z exists in a short form (tRNase ZS) and a long form (tRNase ZL). Prokaryotes have only tRNase ZS, whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase ZL gene encoding both nuclear and mitochondrial forms of tRNase ZL. In contrast, Schizosaccharomyces pombe contains two essential tRNase ZL genes (trz1 and trz2) encoding two tRNase ZL proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3′-end processing, respectively. In addition, trz2 is also involved in generation of the 5′-ends of other mitochondrial RNAs, whose 5′-ends coincide with the 3′-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase ZL activities between two different proteins in S. pombe. The evolution of two tRNase ZL genes and their differential expression in fission yeast may avoid toxic off-target effects.

The fission yeast Schizosaccharomyces pombe has two distinct tRNase ZLs encoded by two different genes and differentially targeted to the nucleus and mitochondria

tRNase Z is the endonuclease that is involved in tRNA 3′-end maturation by removal of the 3′-trailer sequences from tRNA precursors. Most eukaryotes examined to date, including the budding yeast Saccharomyces cerevisiae and humans, have a single long form of tRNase Z (tRNase ZL). In contrast, the fission yeast Schizosaccharomyces pombe contains two candidate tRNase ZLs encoded by the essential genes sptrz1+ and sptrz2+. In the present study, we have expressed recombinant SpTrz1p and SpTrz2p in S. pombe. Both recombinant proteins possess precursor tRNA 3′-endonucleolytic activity in vitro. SpTrz1p localizes to the nucleus and has a simian virus 40 NLS (nuclear localization signal)-like NLS at its N-terminus, which contains four consecutive arginine and lysine residues between residues 208 and 211 that are critical for the NLS function. In contrast, SpTrz2p is a mitochondrial protein with an N-terminal MTS (mitochondrial-targeting signal). High-level overexpression of sptrz1+ has no detectable phenotypes. In contrast, strong overexpression of sptrz2+ is lethal in wild-type cells and results in morphological abnormalities, including swollen and round cells, demonstrating that the correct expression level of sptrz2+ is critical. The present study provides evidence for partitioning of tRNase Z function between two different proteins in S. pombe, although we cannot rule out specialized functions for each protein.

Functional conservation of tRNase ZL among Saccharomyces cerevisiae, Schizosaccharomyces pombe and humans

Although tRNase Z from various organisms was shown to process nuclear tRNA 3′ ends in vitro, only a very limited number of studies have reported its in vivo biological functions. tRNase Z is present in a short form, tRNase ZS, and a long form, tRNase ZL. Unlike Saccharomyces cerevisiae, which contains one tRNase ZL gene (scTRZ1) and humans, which contain one tRNase ZL encoded by the prostate-cancer susceptibility gene ELAC2 and one tRNase ZS, Schizosaccharomyces pombe contains two tRNase ZL genes, designated sptrz1+ and sptrz2+. We report that both sptrz1+ and sptrz2+ are essential for growth. Moreover, sptrz1+ is required for cell viability in the absence of Sla1p, which is thought to be required for endonuclease-mediated maturation of pre-tRNA 3′ ends in yeast. Both scTRZ1 and ELAC2 can complement a temperature-sensitive allele of sptrz1+, sptrz1–1, but not the sptrz1 null mutant, indicating that despite exhibiting species specificity, tRNase ZLs are functionally conserved among S. cerevisiae, S. pombe and humans. Overexpression of sptrz1+, scTRZ1 and ELAC2 can increase suppression of the UGA nonsense mutation ade6–704 through facilitating 3′ end processing of the defective suppressor tRNA that mediates suppression. Our findings reveal that 3′ end processing is a limiting step for defective tRNA maturation and demonstrate that overexpression of sptrz1+, scTRZ1 and ELAC2 can promote defective tRNA 3′ processing in vivo. Our results also support the notion that yeast tRNase ZL is absolutely required for 3′ end processing of at least a few pre-tRNAs even in the absence of Sla1p.