SignificanceConversion of ribonucleotides to the 2′-deoxyribonucleotides required for DNA biosynthesis is catalyzed by ribonucleotide reductases (RNRs) via a free-radical mechanism. Known types of RNRs all depend on redox-active transition metals—manganese, iron, or cobalt—for radical initiation. Pathogenic bacteria are challenged by transition metal sequestration and infliction of oxidative stress by their hosts, and the deployment of multiple RNRs with different metal requirements and radical-initiating oxidants is a known bacterial countermeasure. A class I RNR from two bacterial pathogens completely lacks transition metals in its active state and uses a tyrosine-derived dihydroxyphenylalanine radical as its initiator, embodying a novel tactic to combat transition metal- and oxidant-mediated innate immunity and reinforcing bacterial RNRs as potential antibiotic targets.

S2Materials. Yeast extract, tryptone, and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from DOT Scientific. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycerol, and imidazole, were purchased from Fisher Scientific. Kanamycin was purchased from Teknova. Sodium chloride (NaCl), sodium hydroxide (NaOH), ammonium chloride (NH 4 Cl) and hydrochloric acid (HCl) were purchased from EMD Millipore. Dithiothreitol (DTT) and 1,4-dihydroxynapthalene (NQ) were purchased from Alfa Aeser. Ni II -nitrilotriacetic acid agarose (Ni-NTA) resin and 96-well crystallography screens were purchased from Qiagen. All other chemicals used were purchased from Sigma Aldrich. Oligonucleotide primers were purchased from Integrated DNA Technologies. PCR reagents, restriction enzymes/buffers, and T4 DNA ligase were purchased from New England Biolabs. Escherichia coli (Ec) DH5 α, BL21 (DE3), and the pET-28a(+) vector were purchased from Novagen. All crystallography supplies were purchased from Qiagen and Hampton Research.

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The complement system is a major component of innate immunity and its activation depends on component C3. Complement component C3 is a multidomain protein which can be activated to C3b via any of three activation pathways as the third step in a proteolytic cascade. Once active, C3b continues the activation cascade which eventually leads to creation of a membrane attack complex (MAC) that is able to lyse bacteria and damage host cells. When misregulated, MAC can also attack healthy cells. Due to the complex nature of the activation of the complement system, the more intricate molecular details have not yet been elucidated, but several regulators are known. One of these regulators is thrombomodulin (TM) which is better known for its role in regulating blood clotting. Several in vivo studies have shown that the lectin-like domain of TM interferes with complement activation. Our in vitro work has shown that TM can bind to C3, and we have used hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence assays to begin to understand the effect of TM binding on C3.

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