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.
Highlights
Ribonucleotide reductases (RNRs) are highly important enzymes for all life, as they are solely responsible for the first committed step in the synthesis of the deoxyribonucleoside triphosphate building blocks of DNA [1]
The RNR fold is shared by other enzymes employing glycyl radicals, such as pyruvate formate lyase (PFL), glycerol dehydratase (GD) [16,17], 4-hydroxyphenylacetate decarboxylase (4-HPAD) [18] and benzylsuccinate synthase [19]
One of the latter two cysteines is conserved in class III RNR [9] and both reducing equivalents are provided by the small cosubstrate formate [22] in most systems characterized to date, very recently Trx was shown to be a possible reductant for the NrdD from Neisseria bacilliformis [23]
Summary
Ribonucleotide reductases (RNRs) are highly important enzymes for all life, as they are solely responsible for the first committed step in the synthesis of the deoxyribonucleoside triphosphate (dNTP) building blocks of DNA [1]. The 5’-dAdo radical in turn abstracts an H atom from a glycine residue in a C-terminal, inward-pointing loop in the reductase NrdD [7,8,9] This glycyl radical is stable and can catalyze several dozen cycles before having to be regenerated [10,11].More than five decades of research on RNRs, including the crystal structure determination of several RNRs representing all three classes [9,12,13,14], have led to the insight that, despite often significant differences in sequence and radical generation mechanism, all RNRs are characterized by a 10-stranded α-β barrel fold containing at its heart the “finger loop” [12]. This cysteine is thought to be completely conserved across all RNRs, having been found in the sequences and structures of RNRs from all classes
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