The enzyme ribonucleotide reductase furnishes precursors for the DNA synthesis of all living cells. One of its constituents, the free radical protein, has an unusual alpha-helical structure. There are two iron centres that are about 25 A apart in the dimeric molecule. Tyrosine 122, which harbours the stable free radical necessary for the activity of ribonucleotide reductase, is buried inside the protein and is located 5 A from the closest iron atom.

Binding of substrate at the active site of the enzyme is structurally regulated in two ways: binding of the correct substrate is regulated by the binding of allosteric effectors and binding of the actual substrate occurs primarily when the active-site cysteines are reduced. One of the loops stabilized upon binding of dTTP participates in the formation of the substrate-binding site through direct interaction with the nucleotide base. The general allosteric effector site, located far from the active site, appears to regulate subunit interactions within the holoenzyme.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox‐active S‐S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33‐34, 75‐76 and 91‐93 in E. coli thioredoxin; 15‐16, 65‐66 and 76‐78 in T4 thioredoxin and 12‐13, 59‐60 and 69‐71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S‐S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.

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The small subunit of iron‐dependent ribonucleotide reductases contains a stable organic free radical, which is essential for enzyme activity and which is localized to a tyrosine residue. Tyrosine‐122 in the B2 subunit of Escherichia coli ribonucleotide reductase has been changed into a phenylalanine. The mutation was introduced with oligonucleotide‐directed mutagenesis in an M13 recombinant and verified by DNA sequencing. Purified native and mutant B2 protein were found to have the same size, iron content and iron‐related absorption spectrum. The sole difference observed is that the mutant protein lacks tyrosyl radical and enzymatic activity. These results identify Tyr122 of E. coli protein B2 as the tyrosyl radical residue. An expression vector was constructed for manipulation and expression of ribonucleotide reductase subunits. It contains the entire nrd operon with its own promoter in a 2.3‐kb fragment from pBR322. Both the B1 and the B2 subunits were expressed at a 25‐35 times higher level as compared to the host strain.

During anaerobic growth of Escherichia coli an oxygen-sensitive ribonucleoside-triphosphate reductase, different from the aerobic ribonucleoside diphosphate-reductase (EC 1.17.4.1), produces the deoxyribonucleoside triphosphates required for DNA replication. The gene for the anaerobic enzyme has now been cloned and was found to contain a 2136-nucleotide coding region, corresponding to 712 amino acid residues, and an Fnr binding site 228 base pairs upstream of the initiator ATG. The deduced amino acid sequence shows 72% identity to a gene of coliphage T4, sunY, hitherto of unknown function, suggesting that the virus codes for its own anaerobic reductase. The location of an organic free radical formed during activation of the bacterial anaerobic reductase is proposed to be on Gly-681, since the pentapeptide RVCGY at positions 678-682 shows a striking similarity to the C-terminal sequence, RVSGY, of pyruvate formate-lyase. During activation of the anaerobically induced pyruvate formate-lyase,

Ribonucleotide reductase (RNR) catalyzes the only known de-novo pathway for production of all four deoxyribonucleotides required for DNA synthesis1,2. It is essential for all organisms with DNA as genetic material and a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a di-nuclear metal site has been viewed as a requirement for generating and stabilizing a catalytic radical, essential for RNR activity5,6,7. Here, we describe a new group of RNR proteins in Mollicutes, including Mycoplasma pathogens, which possesses a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal an unprecedented stable DOPA radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement of a dinuclear metal site in aerobic RNR. The metal-independent radical compels completely novel mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. Conceivably, this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms encoding this type of RNR are involved in diseases of the respiratory, urinary and genital tracts, some with developing resistance to antibiotics. Further characterization of this novel RNR family and its mechanism for cofactor generation will provide insight into new enzymatic chemistry and be of value to devise strategies to combat the pathogens that utilize it. We propose that the new RNR subclass is denoted class Ie.

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