Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.

Rhenium(V) oxo complexes of general formula [ReO(OMe)(N^N)Cl2], where N^N = 4,7-diphenyl-1,10-phenanthroline, 1, or 3,4,7,8-tetramethyl-1,10-phenanthroline, 2, effectively kill cancer cells by triggering necroptsosis, a non-apoptotic form of cell death. Both complexes evoke necrosome (RIP1-RIP3)-dependent intracellular ROS production and propidium iodide uptake. The complexes also induce mitochondrial membrane potential depletion, a possible downstream effect of ROS production. Apparently, 1 and 2 are the first rhenium complexes to evoke cellular events consistent with programmed necrosis in cancer cells. Furthermore, 1 and 2 display low acute toxicity in C57BL/6 mice and reasonable stability in fresh human blood.

Bacteria account for 1000-fold more biomass than humans. They vary widely in shape and size. The morphological diversity of bacteria is due largely to the different peptidoglycan-based cell wall structures that encase bacterial cells. Although the basic structure of peptidoglycan is highly conserved, consisting of long glycan strands that are cross-linked by short peptide chains, the mature cell wall is chemically diverse. Peptidoglycan hydrolases and cell wall–tailoring enzymes that regulate glycan strand length, the degree of cross-linking, and the addition of other modifications to peptidoglycan are central in determining the final architecture of the bacterial cell wall. Historically, it has been difficult to biochemically characterize these enzymes that act on peptidoglycan because suitable peptidoglycan substrates were inaccessible. In this review, we discuss fundamental aspects of bacterial cell wall synthesis, describe the regulation and diverse biochemical and functional activities of peptidoglycan hydrolases, and highlight recently developed methods to make and label defined peptidoglycan substrates. We also review how access to these substrates has now enabled biochemical studies that deepen our understanding of how bacterial cell wall enzymes cooperate to build a mature cell wall. Such improved understanding is critical to the development of new antibiotics that disrupt cell wall biogenesis, a process essential to the survival of bacteria.

The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.