The Zn center of the anaerobic ribonucleotide reductase from E. coli

Strict and facultative anaerobes depend on a class III ribonucleotide reductase for their growth. These enzymes are the sole cellular catalysts for de novo biosynthesis of the deoxyribonucleotides needed for DNA chain elongation and repair. In its active form, the class III ribonucleotide reductase from Escherichia coli contains a free radical located on the G681 residue which is essential for the activation of the ribonucleotide substrate toward its reduction. The 3D structure of the homologous enzyme from bacteriophage T4 has revealed the presence of a metal center bound to four conserved cysteine residues. In this report we identify the metal of the E. coli enzyme as Zn. We show that the presence of Zn in this site protects the protein from proteolysis and prevents the formation of disulfide bridges within it. Finally, we show with the fully Zn-loaded reductase that thioredoxin or small thiols are dispensable for the formation of the glycyl radical. However, they are necessary for obtaining high turnover numbers, suggesting that they intervene in radical transfer steps subsequent to the formation of the glycyl radical.     

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate

Dihydrofolate reductase (DHFR) is a ubiquitous enzyme involved in major biological process, including DNA synthesis and cancer inhibition, and its modulation is the object of extensive structural, kinetic, and pharmacological studies. In particular, earlier studies showed that green tea catechins are powerful inhibitors of bovine liver and chicken liver DHFR. In this article, we report the results of inhibition kinetics for the enzyme from another source (DHFR from E. coli) exerted by (-)-epigallocatechingallate (EGCG). Using different analytical techniques, we reported that EGCG acts as a bisubstrate inhibitor on the bacterial DHFR. Moreover, the combined approach of biosensor, kinetic, and molecular modelling analysis disclosed the ability of EGCG to bind to the enzyme both on substrate (DHF) and cofactor (NADPH) site. Collectively, our data have confirmed the selectivity of antifolate compounds with respect to the different source of enzyme (bacterial or mammalian DHFR) and the possible role of tea catechins as chemopreventive agents.     

Evidence for a new ribonucleotide reductase in anaerobic E. coli

E. coli conditional iron-containing ribonucleotide reductase (Fe-RR) mutant and wild type strains grew anaerobically under conditions when Fe-RR was absent or inhibited. Furthermore, a B12-independent, hydroxyurea-resistant RR activity, unaffected by monoclonal antibodies against either subunit B1 or B2 of Fe-RR, was partially purified from anaerobically grown mutant and wild-type E. coli. These findings indicate that E. coli has a second RR representative of a new class of RRs and that this is the first report where both in vivo and in vitro evidence is presented. It is probable that other facultative anaerobes also have two different RRs such that an optimal supply of deoxyribonucleotides is maintained under all growth conditions.     

Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and G?nzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).     

Pre-steady-state and steady-state kinetic analysis of E. coli class I ribonucleotide reductase

E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.     

Activation of class III ribonucleotide reductase from E. coli. The electron transfer from the iron-sulfur center to S-adenosylmethionine

The anaerobic ribonucleotide reductase (ARR) from E. coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals. ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a [4Fe-4S]-containing activating enzyme (beta) and AdoMet under reducing conditions. Here we show that the EPR-active S = 1/2 reduced [4Fe-4S]+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical. These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet. Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT. However, in that case, 2 equiv of methionine per reduced cluster was formed. This unusual stoichiometry and combined EPR and M?ssbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active [3Fe-4S] intermediate clusters.     

The tyrosine free radical in ribonucleotide reductase from Escherichia coli.

One of the two nonidentical subunits of ribonucleotide reductase from Escherichia coli, protein B2, contains an organic free radical required for enzyme activity. Earlier isotope subtitution experiments (Sj?berg, B.-M., Reichard, P. Gr?slund, A., and Ehrenberg, A. (1977) J. Biol. Chem. 252, 536-541) demonstrated that the radical was localized to a tyrosine residue of the enzyme and suggested that the spin density of the radical was centered at the methylene carbon of tyrosine. However, additional isotope substitution experiments now show that the spin density of the radical must be delocalized over the aromatic ring of the tyrosine residue.     

Nature of the free radical in ribonucleotide reductase from Escherichia coli.

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site of the enzyme is made up from both subunits. Protein B2 contributes inter alia an organic free radical which gives a characteristic EPR signal. This radical was now located by isotope substitution experiments to the beta position of a tyrosine residue. The EPR spectrum of protein B2 from bacteria grown in a completely deuterated medium was drastically changed. The change was reversed by the addition of other protonated amino acids. The involvement in radical formation of the beta position of tyrosine was demonstrated from EPR spectra of protein B2 from bacteria grown in the presence of specifically deuterated tyrosine.     

The iron center in ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase from Escherichia coli consists of two nonidentical subunits, proteins B1 and B2. The active site is made up from both subunits. Protein B2 contains 2 iron atoms and a tyrosyl-free radical, which are essential for the enzymatic activity. The paramagnetic susceptibility of protein B2 has been measured over the temperature range 30-200 K. A deviation from the Curie law is observed at high temperatures, consistent with a structure of an antiferromagnetically coupled pair of high spin Fe(III) with an exchange coupling -J = 108(-20)+25 cm-1. Electronic spectra are resolved into components from the iron center and the radical. A band at 600 nm is clearly identified and shown to have contributions from both components. The electronic absorptions of the tyrosyl radical of protein B2 are closely similar to those reported for phenoxy radicals of tyrosine and tritertiary butyl phenol. Determinations by EPR of the amount of free radical suggest the possibility of more than one radical per active protein B2 molecule. Reconstitution of the active site from apoprotein B2 and Fe(II) is only observed in the presence of oxygen. With Fe(III), no reconstitution is obtained. The additional physical data on the iron center of protein B2 strengthen the analogy with oxidized forms of hemerythrin. The most likely structure is an antiferromagnetically coupled pair of high spin Fe(III), possibly with a bridging oxo-group.     

Irreversible inhibition of ribonucleotide reductase from Ehrlich tumor cells by a modulator analog

Periodate-oxidized ATP (ATP-PI) was prepared and studied as a modulator analog of ATP in the ribonucleotide reductase system from Ehrlich tumor cells. ATP-PI could not replace ATP as an activator of CDP reduction, but was found to be an effective inhibitor of both CDP and ADP reduction. The inhibition was time dependent with 1 μM ATP-PI causing 100% inhibition after 20 hrs. The inhibition was shown to be irreversible by the Ackermann-Potter plot and enzyme activity was not restored by passage of the ATP-PI-treated enzyme over a Sephadex G-25 column. ¹⁴C-ATP-PI eluted with the protein peak on Sephadex G-25 chromatography.     

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).     

Iron-sulfur interconversions in the anaerobic ribonucleotide reductase from Escherichia coli

The anaerobic ribonucleotide reductase from Escherichia coli contains an iron-sulfur cluster which, in the reduced [4Fe-4S]⁺ form, serves to reduce S-adenosylmethionine and to generate a catalytically essential glycyl radical. The reaction of the reduced cluster with oxygen was studied by UV-visible, EPR, NMR, and Mössbauer spectroscopies. The [4Fe-4S]⁺ form is shown to be extremely sensitive to oxygen and converted to [4Fe-4S]²⁺, [3Fe-4S]^+/0, and to the stable [2Fe-2S]²⁺ form. It is remarkable that the oxidized protein retains full activity. This is probably due to the fact that during reduction, required for activity, the iron atoms, from 2Fe and 3Fe clusters, readily reassemble to generate an active [4Fe-4S] center. This property is discussed as a possible protective mechanism of the enzyme during transient exposure to air. Futhermore, the [2Fe-2S] form of the protein can be converted into a [3Fe-4S] form during chromatography on dATP-Sepharose, explaining why previous preparations of the enzyme were shown to contain large amounts of such a 3Fe cluster. This is the first report of a 2Fe to 3Fe cluster conversion.     

Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (F(n)Ys) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1-353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the F(n)Y at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the F(n)Y at residue 356. The procedure to generate 100 mg quantities of Y356F(n)Y-R2 takes 3-4 months.     

Mutant R1 proteins from Escherichia coli class Ia ribonucleotide reductase with altered responses to dATP inhibition

Aerobic ribonucleotide reductase from Escherichia coli regulates its level of activity by binding of effectors to an allosteric site in R1, located to the proposed interaction area of the two proteins that comprise the class I enzyme. Activity is increased by ATP binding and decreased by dATP binding. To study the mechanism governing this regulation, we have constructed three R1 proteins with mutations at His-59 in the activity site and one R1 protein with a mutation at His-88 close to the activity site and compared their allosteric behavior to that of the wild type R1 protein. All mutant proteins retained about 70% of wild type enzymatic activity. We found that if residue His-59 was replaced with alanine or asparagine, the enzyme lost its normal response to the inhibitory effect of dATP, whereas the enzyme with a glutamine still managed to elicit a normal response. We saw a similar result if residue His-88, which is proposed to hydrogen-bond to His-59, was replaced with alanine. Nucleotide binding experiments ruled out the possibility that the effect is due to an inability of the mutant proteins to bind effector since little difference in binding constants was observed for wild type and mutant proteins. Instead, the interaction between proteins R1 and R2 was perturbed in the mutant proteins. We propose that His-59 is important in the allosteric effect triggered by dATP binding, that the conserved hydrogen bond between His-59 and His-88 is important for the communication of the allosteric effect, and that this effect is exerted on the R1/R2 interaction.     

Studies on a membrane-bound and solubilized ribonucleotide reductase preparation from Escherichia coli TAU-.

Ribonucleotide reductase has been shown to be associated with the DNA-membrane complex in Escherichia coli TAU- cells. The membrane-bound enzyme has been released in a soluble form using a combined treatment of 1% sarcosyl (pH 8.0) and 1% sodium deoxycholate (pH 6.5). Allotropic differences in the modulatory effects of ATP, Mg2+, EDTA and dithiothreitol on the membrane-bound and solubilized enzyme activity are discussed.     

Redox studies of subunit interactivity in aerobic ribonucleotide reductase from Escherichia coli

Ribonucleotide reductase is a heterodimeric (alpha(2)beta(2)) allosteric enzyme that catalyzes the conversion of ribonucleotides to deoxyribonucleotides, an essential step in DNA biosynthesis and repair. In the enzymatically active form aerobic Escherichia coli ribonucleotide reductase is a complex of homodimeric R1 and R2 proteins. We use electrochemical studies of the dinuclear center to clarify the interplay of subunit interaction, the binding of allosteric effectors and substrate selectivity. Our studies show for the first time that electrochemical reduction of active R2 generates a distinct Met form of the diiron cluster, with a midpoint potential (-163 +/- 3 mV) different from that of R2(Met) produced by hydroxyurea (-115 +/- 2 mV). The redox potentials of both Met forms experience negative shifts when measured in the presence of R1, becoming -223 +/- 6 and -226 +/- 3 mV, respectively, demonstrating that R1-triggered conformational changes favor one configuration of the diiron cluster. We show that the association of a substrate analog and specificity effector (dGDP/dTTP or GMP/dTTP) with R1 regulates the redox properties of the diiron centers in R2. Their midpoint potential in the complex shifts to -192 +/- 2 mV for dGDP/dTTP and to -203 +/- 3 mV for GMP/dTTP. In contrast, reduction potential measurements show that the diiron cluster is not affected by ATP (0.35-1.45 mm) and dATP (0.3-0.6 mm) binding to R1. Binding of these effectors to the R1-R2 complex does not perturb the normal docking modes between R1 and R2 as similar redox shifts are observed for ATP or dATP associated with the R1-R2 complex.