Essential for DNA biosynthesis and repair, ribonucleotide reductases (RNRs) convert ribonucleotides to deoxyribonucleotides via radical-based chemistry. Although long known that allosteric regulation of RNR activity is vital for cell health, the molecular basis of this regulation has been enigmatic, largely due to a lack of structural information about how the catalytic subunit (α 2 ) and the radical-generation subunit (β 2 ) interact. Here we present the first structure of a complex between α 2 and β 2 subunits for the prototypic RNR from Escherichia coli. Using four techniques (small-angle X-ray scattering, X-ray crystallography, electron microscopy, and analytical ultracentrifugation), we describe an unprecedented α 4 β 4 ring-like structure in the presence of the negative activity effector dATP and provide structural support for an active α 2 β 2 configuration. We demonstrate that, under physiological conditions, E. coli RNR exists as a mixture of transient α 2 β 2 and α 4 β 4 species whose distributions are modulated by allosteric effectors. We further show that this interconversion between α 2 β 2 and α 4 β 4 entails dramatic subunit rearrangements, providing a stunning molecular explanation for the allosteric regulation of RNR activity in E. coli.allostery | protein-protein interactions | conformational equilibria | nucleotide metabolism I mportant targets of anticancer and antiviral drugs, ribonucleotide reductases (RNRs) are classified by the metallocofactor used to generate a thiyl radical (1) that initiates reduction of ribonucleotides to deoxyribonucleotides (2, 3). Class Ia RNRs are found in all eukaryotes and many aerobic bacteria, with the Escherichia coli enzyme serving as the prototype. These RNRs reduce ribonucleoside 5′-diphosphates and are composed of two homodimeric subunits: α 2 and β 2 (Fig. 1A). The α 2 subunit contains the active site, where ribonucleotide reduction occurs, and two types of allosteric effector binding sites (4, 5). One effector site tunes the specificity for all four ribonucleotide substrates in response to intracellular levels of deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP) and ATP (6, 7) such that balanced pools of deoxyribonucleotides are maintained (8). The second effector site controls the rate of reduction, binding ATP to turn the enzyme on or dATP to turn it off (4, 6). This activity site is housed in an N-terminal cone domain (5, 9) and provides a means for negative feedback regulation to safeguard against cytotoxic elevation of deoxyribonucleotide levels (2, 3, 10). The β 2 subunit harbors the essential diferric-tyrosyl radical (Y122• in E. coli) cofactor (11) that initiates radical chemistry.Active RNR has long been proposed to be a transient α 2 β 2 complex (Fig. 1B) with enhanced subunit affinity upon binding of substrates and effectors (12-15). For each turnover, α 2 , β 2 , substrate and effector must interact, triggering long-range proton-coupled electron transfer (PCET) reducing Y122• in β 2 and oxidizing C439 to a thiyl radical in the active s...

Genome sequencing of environmental bacteria allows identification of biosynthetic gene clusters encoding unusual combinations of enzymes that produce unknown natural products. We identified a pathway in which a ribosomally synthesized small peptide serves as a scaffold for nonribosomal peptide extension and chemical modification. Amino acids are transferred to the carboxyl terminus of the peptide through adenosine triphosphate and amino acyl-tRNA–dependent chemistry that is independent of the ribosome. Oxidative rearrangement, carboxymethylation, and proteolysis of a terminal cysteine yields an amino acid–derived small molecule. Microcrystal electron diffraction demonstrates that the resulting product is isosteric to glutamate. We show that a similar peptide extension is used during the biosynthesis of the ammosamides, which are cytotoxic pyrroloquinoline alkaloids. These results suggest an alternative paradigm for biosynthesis of amino acid–derived natural products.

Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction, and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O, and C-N bond breaking and formation steps that are otherwise challenging for non-radical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.

Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell.DOI: http://dx.doi.org/10.7554/eLife.07141.001