Structures of Class Id Ribonucleotide Reductase Catalytic Subunits Reveal a Minimal Architecture for Deoxynucleotide Biosynthesis

Abstract

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.

Solution Structure of the dATP-Inactivated Class I Ribonucleotide Reductase From Leeuwenhoekiella blandensis by SAXS and Cryo-Electron Microscopy

“…No crystal structure of LbNrdA has been determined to date. A reliable homology model for residues 40–596 was prepared using the SwissModel server with the dimeric crystal structure of Actinobacillus ureae (PDB ID 6DQX) ( Rose et al, 2019 ) as template. This NrdA has 59% sequence identity to LbNrdA over 92% of the length of the latter.…”

“…Comparative statistics are shown in Supplementary Table 1 . The Phyre2 and I-TASSER models had poorer overall quality but predicted a structure for the N-terminal 39 residues based on weak homology to three helices in the “connector domain” ( Parker et al, 2018 ; Rose et al, 2019 ) that falls between the ATP-cone and the core in many class Ia RNRs. However, this three-helix bundle packed against the core of the protein such that its hydrophobic core enclosed three charged residues from the surface of the core: Glu69, Arg72, and Asp76.…”

“…A3XHF8, gene MED217_17,130). The best template found was the 1.76 Å resolution crystal structure of the class Id NrdA from Actinobacillus ureae (PDB ID 6DQX) ( Rose et al, 2019 ), which has 59% sequence identity to LbNrdA over 92% of the length of LbNrdA. In addition, models were generated using the Phyre2 ( Kelley et al, 2015 ) and I-TASSER ( Yang et al, 2015 ) servers.…”

“…In eukaryotic RNR, three dimers of NrdA form a ring-shaped α 6 hexamer ( Fairman et al, 2011 ; Brignole et al, 2018 ) to which one β 2 dimer can attach ( Rofougaran et al, 2006 ; Fairman et al, 2011 ; Ando et al, 2016 ), though the interactions with NrdB and how they break the PCET chain are not fully structurally characterized for this system. More recently, higher order oligomers such as helices have also been proposed both for class Ib RNRs with partial ATP cones ( Thomas et al, 2019 ) and class Id RNRs lacking ATP cones ( Rose et al, 2019 ).…”

Formation of Unstable and very Reactive Chemical Species Catalyzed by Metalloenzymes: A Mechanistic Overview

“…The most studied ones are from class I and class II. Class I RNRs are oxygen-dependent enzymes and are present in both eukaryotes and eubacteria and contain a binuclear metal cluster formed by Fe or Mn [153,154,155]. Class II RNRs, found in both aerobic and anaerobic microbes, are oxygen-independent and use a cobalt-containing cobalamin (vitamin B12) cofactor [156,157].…”