The Crystal Structure of Thermotoga maritima Class III Ribonucleotide Reductase Lacks a Radical Cysteine Pre-Positioned in the Active Site

Abstract

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, the building blocks for DNA synthesis, and are found in all but a few organisms. RNRs use radical chemistry to catalyze the reduction reaction. Despite RNR having evolved several mechanisms for generation of different kinds of essential radicals across a large evolutionary time frame, this initial radical is normally always channelled to a strictly conserved cysteine residue directly adjacent to the substrate for initiation of substrate reduction, and this cysteine has been found in the structures of all RNRs solved to date. We present the crystal structure of an anaerobic RNR from the extreme thermophile Thermotoga maritima (tmNrdD), alone and in several complexes, including with the allosteric effector dATP and its cognate substrate CTP. In the crystal structure of the enzyme as purified, tmNrdD lacks a cysteine for radical transfer to the substrate pre-positioned in the active site. Nevertheless activity assays using anaerobic cell extracts from T. maritima demonstrate that the class III RNR is enzymatically active. Other genetic and microbiological evidence is summarized indicating that the enzyme is important for T. maritima. Mutation of either of two cysteine residues in a disordered loop far from the active site results in inactive enzyme. We discuss the possible mechanisms for radical initiation of substrate reduction given the collected evidence from the crystal structure, our activity assays and other published work. Taken together, the results suggest either that initiation of substrate reduction may involve unprecedented conformational changes in the enzyme to bring one of these cysteine residues to the expected position, or that alternative routes for initiation of the RNR reduction reaction may exist. Finally, we present a phylogenetic analysis showing that the structure of tmNrdD is representative of a new RNR subclass IIIh, present in all Thermotoga species plus a wider group of bacteria from the distantly related phyla Firmicutes, Bacteroidetes and Proteobacteria.

New tricks for the glycyl radical enzyme family

“…Until recently it was thought that all class III RNRs used formate as the reductant for ribonucleotide reduction and utilized a conserved set of active site residues, but recent phylogenetic analyses on class III RNRs (Wei et al , 2014a, Aurelius et al , 2015) and a metabolic pathway analysis (Wei et al , 2015) have suggested that this is not the case. Currently, it appears that there are at least three subclasses of NrdDs, which can be distinguished by the presence or absence of 3–4 catalytic residues and the requirement for formate as the reductant.…”

The Origin and Evolution of Ribonucleotide Reduction

“… (1) Two recent independently solved structures of Thermotoga maritima class III RNR lack a suitably positioned cysteine [ 45 , 46 ]. Further work is required to determine the mechanistic details of the protein.…”

“…Adding further mystery to the class III reaction mechanism, even the unanimous presence of the radical cysteine has been called into question by two recent independent crystal structures of T. maritima class III RNR [ 45 , 46 ]. At the position of the canonical radical cysteine is an isoleucine in both structures.…”

“…The cysteine that is believed to be the radical instigator based on sequence alignments is positioned outside the active site in the crystal structures. Biochemical evidence based on mutation of this cysteine and its neighbor suggest strongly that these residues are found in the active site during catalysis [ 45 ], but the conformational changes required to position the radical cysteine near the substrate would be unprecedented in RNRs, so the implications of these structures have still to be unraveled.…”

Ribonucleotide Reductases from Bifidobacteria Contain Multiple Conserved Indels Distinguishing Them from All Other Organisms: In Silico Analysis of the Possible Role of a 43 aa Bifidobacteria-Specific Insert in the Class III RNR Homolog

“…In all three RNRs, allosteric regulation involves binding of the dNDP/dNTP products at a 4-helix bundle, involving two helices from each monomeric subunit, at the dimer interface of the enzyme ( Uhlin and Eklund, 1994 ; Larsson et al, 2001 ). The allosteric regulation causes conformational changes at a highly conserved 10 stranded α/β barrel where the active site “finger loop” structure resides in its center, or is brought to its center upon activation ( Aurelius et al, 2015 ).…”