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.

Human DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is.

Resistance to β-lactams is one of the most serious problems associated with Gram-negative infections. β-Lactamases are able to hydrolyze β-lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo-β-lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown. We discovered PNGM-1, the novel subclass B3 MBL, in deep-sea sediments that predate the antibiotic era. Here, our phylogenetic analysis suggests that PNGM-1 yields insights into the evolutionary origin of subclass B3 MBLs. We reveal the structural similarities between tRNase Zs and PNGM-1, and demonstrate that PNGM-1 has both MBL and tRNase Z activities, 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 MBLs and tRNase Zs. These comparisons revealed that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity.