Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.     

(Mu-1,2-peroxo)diiron(III/III) complex as a precursor to the diiron(III/IV) intermediate X in the assembly of the iron-radical cofactor of ribonucleotide reductase from mouse

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and M?ssbauer spectroscopies have been used to obtain evidence for the intermediacy of a (mu-1,2-peroxo)diiron(III/III) complex on the pathway to the tyrosyl radical and (mu-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to approximately 0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (mu-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O2 activation. The detection of the (mu-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Delta9 desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.     

Cloning and characterization of ribonucleotide reductase from Chlamydia trachomatis

In all organisms the deoxyribonucleotide precursors required for DNA synthesis are synthesized from ribonucleotides, a reaction catalyzed by ribonucleotide reductase. In a previous study we showed that Chlamydia trachomatis growth was inhibited by hydroxyurea, an inhibitor of ribonucleotide reductase, and a mutant resistant to the cytotoxic effects of the drug was isolated. Here we report the cloning, expression, and purification of the R1 and R2 subunits of the C. trachomatis ribonucleotide reductase. In comparison with other ribonucleotide reductases, the primary sequence of protein R1 has an extended amino terminus, and the R2 protein has a phenylalanine where the essential tyrosine is normally located. Despite its unusual primary structure, the recombinant enzyme catalyzes the reduction of CDP to dCDP. Results from deletion mutagenesis experiments indicate that while the extended amino terminus of the R1 protein is not required for enzyme activity, it is needed for allosteric inhibition mediated by dATP. Results with site-directed mutants of protein R2 suggest that the essential tyrosine is situated two amino acids downstream of its normal location. Finally, Western blot analysis show that the hydroxyurea-resistant mutant C. trachomatis isolate overexpresses both subunits of ribonucleotide reductase. At the genetic level, compared with wild type C. trachomatis, the resistant isolate has a single base mutation just upstream of the ATG start codon of the R2 protein. The possibility that this mutation affects translational efficiency is discussed.     

Rapid and quantitative activation of Chlamydia trachomatis ribonucleotide reductase by hydrogen peroxide

We recently showed that the class Ic ribonucleotide reductase (RNR) from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor in its R2 subunit to initiate catalysis [Jiang, W., Yun, D., Saleh, L., Barr, E. W., Xing, G., Hoffart, L. M., Maslak, M.-A., Krebs, C., and Bollinger, J. M., Jr. (2007) Science 316, 1188-1191]. The Mn (IV) site of the novel cofactor functionally replaces the tyrosyl radical used by conventional class I RNRs to initiate substrate radical production. As a first step in evaluating the hypothesis that the use of the alternative cofactor could make the RNR more robust to reactive oxygen and nitrogen species [RO(N)S] produced by the host's immune system [H?gbom, M., Stenmark, P., Voevodskaya, N., McClarty, G., Gr?slund, A., and Nordlund, P. (2004) Science 305, 245-248], we have examined the reactivities of three stable redox states of the Mn/Fe cluster (Mn (II)/Fe (II), Mn (III)/Fe (III), and Mn (IV)/Fe (III)) toward hydrogen peroxide. Not only is the activity of the Mn (IV)/Fe (III)-R2 intermediate stable to prolonged (>1 h) incubations with as much as 5 mM H 2O 2, but both the fully reduced (Mn (II)/Fe (II)) and one-electron-reduced (Mn (III)/Fe (III)) forms of the protein are also efficiently activated by H 2O 2. The Mn (III)/Fe (III)-R2 species reacts with a second-order rate constant of 8 +/- 1 M (-1) s (-1) to yield the Mn (IV)/Fe (IV)-R2 intermediate previously observed in the reaction of Mn (II)/Fe (II)-R2 with O 2 [Jiang, W., Hoffart, L. M., Krebs, C., and Bollinger, J. M., Jr. (2007) Biochemistry 46, 8709-8716]. As previously observed, the intermediate decays by reduction of the Fe site to the active Mn (IV)/Fe (III)-R2 complex. The reaction of the Mn (II)/Fe (II)-R2 species with H 2O 2 proceeds in three resolved steps: sequential oxidation to Mn (III)/Fe (III)-R2 ( k = 1.7 +/- 0.3 mM (-1) s (-1)) and Mn (IV)/Fe (IV)-R2, followed by decay of the intermediate to the active Mn (IV)/Fe (III)-R2 product. The efficient reaction of both reduced forms with H 2O 2 contrasts with previous observations on the conventional class I RNR from Escherichia coli, which is efficiently converted from the fully reduced (Fe 2 (II/II)) to the "met" (Fe 2 (III/III)) form [Gerez, C., and Fontecave, M. (1992) Biochemistry 31, 780-786] but is then only very inefficiently converted from the met to the active (Fe 2 (III/III)-Y (*)) form [Sahlin, M., Sj?berg, B.-M., Backes, G., Loehr, T., and Sanders-Loehr, J. (1990) Biochem. Biophys. Res. Commun. 167, 813-818].     

Class Id ribonucleotide reductase utilizes a Mn2(IV,III) cofactor and undergoes large conformational changes on metal loading

JBIC Journal of Biological Inorganic Chemistry - Outside of the photosynthetic machinery, high-valent manganese cofactors are rare in biology. It was proposed that a recently discovered subclass of...     

Escherichia coli class Ib ribonucleotide reductase contains a dimanganese(III)-tyrosyl radical cofactor in vivo

Escherichia coli class Ib ribonucleotide reductase (RNR) converts nucleoside 5'-diphosphates to deoxynucleoside 5'-diphosphates in iron-limited and oxidative stress conditions. We have recently demonstrated in vitro that this RNR is active with both diferric-tyrosyl radical (Fe(III)(2)-Y(?)) and dimanganese(III)-Y(?) (Mn(III)(2)-Y(?)) cofactors in the β2 subunit, NrdF [Cotruvo, J. A., Jr., and Stubbe, J. (2010) Biochemistry 49, 1297-1309]. Here we demonstrate, by purification of this protein from its endogenous levels in an E. coli strain deficient in its five known iron uptake pathways and grown under iron-limited conditions, that the Mn(III)(2)-Y(?) cofactor is assembled in vivo. This is the first definitive determination of the active cofactor of a class Ib RNR purified from its native organism without overexpression. From 88 g of cell paste, 150 μg of NrdF was isolated with ～95% purity, with 0.2 Y(?)/β2, 0.9 Mn/β2, and a specific activity of 720 nmol min(-1) mg(-1). Under these conditions, the class Ib RNR is the primary active RNR in the cell. Our results strongly suggest that E. coli NrdF is an obligate manganese protein in vivo and that the Mn(III)(2)-Y(?) cofactor assembly pathway we have identified in vitro involving the flavodoxin-like protein NrdI, present inside the cell at catalytic levels, is operative in vivo.     

Oxygen cleavage with manganese and iron in ribonucleotide reductase from <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

The oxygen cleavage in Chlamydia trachomatis ribonucleotide reductase (RNR) has been studied using B3LYP* hybrid density functional theory. Class Ic C. trachomatis RNR lacks the radical-bearing tyrosine, crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)–Tyr(rad) active state, C. trachomatis RNR has a mixed Mn(IV)Fe(III) metal center in subunit II (R2). A mixed MnFe metal center has never been observed as a radical cofactor before. The active state is generated by reductive oxygen cleavage at the metal site. On the basis of calculated barriers for oxygen cleavage in C. trachomatis R2 and R2 from Escherichia coli with a diiron, a mixed manganese–iron, and a dimanganese center, conclusions can be drawn about the effect of changing metals in R2. The oxygen cleavage is found to be governed by two factors: the redox potentials of the metals and the relative stability of the different peroxides. Mn(IV) has higher stability than Fe(IV), and the barrier is therefore lower with a mixed metal center than with a diiron center. With a dimanganese center, an asymmetric peroxide is more stable than the symmetric peroxide, and the barrier therefore becomes too high. Calculated proton-coupled redox potentials are compared to identify three possible R2 active states, the Fe(III)Fe(III)–Tyr(rad) state, the Mn(IV)Fe(III) state, and the Mn(IV)Mn(IV) state. A tentative energy profile of the thermodynamics of the radical transfer from R2 to subunit I is constructed to illustrate how the stability of the active states can be understood from a thermodynamical point of view.     

Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B. cereus reconstituted with iron or alternatively with manganese ions, then subsequently reacted with molecular oxygen to generate two tyrosyl-radicals. The two similar X-band EPR spectra did not change significantly over 4 to 50 K. From the 285 GHz EPR spectrum of the iron form, a g ₁-value of 2.0090 for the tyrosyl radical was extracted. This g ₁-value is similar to that observed in class Ia E. coli R2 and class Ib R2Fs with iron-oxygen cluster, suggesting the absence of hydrogen bond to the phenoxyl group. This was confirmed by resonance Raman spectroscopy, where the stretching vibration associated to the radical (C-O, ν_7a = 1500 cm⁻¹) was found to be insensitive to deuterium-oxide exchange. Additionally, the ¹⁸O-sensitive Fe-O-Fe symmetric stretching (483 cm⁻¹) of the metallo-cofactor was also insensitive to deuterium-oxide exchange indicating no hydrogen bonding to the di-iron-oxygen cluster, and thus, different from mouse R2 with a hydrogen bonded cluster. The HF-EPR spectrum of the manganese reconstituted RNR R2F gave a g ₁-value of ∼2.0094. The tyrosyl radical microwave power saturation behavior of the iron-oxygen cluster form was as observed in class Ia R2, with diamagnetic di-ferric cluster ground state, while the properties of the manganese reconstituted form indicated a magnetic ground state of the manganese-cluster. The recent activity measurements (Crona et al., (2011) J Biol Chem 286: 33053–33060) indicates that both the manganese and iron reconstituted RNR R2F could be functional. The manganese form might be very important, as it has 8 times higher activity.     

Substitution of manganese for iron in ribonucleotide reductase from Escherichia coli. Spectroscopic and crystallographic characterization.

Each polypeptide chain of protein R2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a stable tyrosyl radical and two antiferromagnetically coupled oxo-bridged ferric ions. A refined structure of R2 has been recently obtained. R2 can be converted into apoR2 by chelating out the metal cofactor and scavenging the radical. This study shows that apoR2 has a very strong affinity for four stable Mn2+ ions. The manganese-containing form of R2, named Mn-R2, has been studied by EPR spectroscopy and x-ray crystallography. It contains two binuclear manganese clusters in which the two manganese ions occupy the natural iron-binding sites and are only bridged by carboxylates from glutamates 115 and 238. This in turn explains why the spin-exchange interaction between the two ions is very weak and why Mn-R2 is EPR active. Mn-R2 could provide a model for the native diferrous form of protein R2, and a detailed molecular mechanism for the reduction of the iron center of protein R2 is proposed.     

Regulation of ribonucleotide reductase in response to iron deficiency

Ribonucleotide reductase (RNR) is an essential enzyme required for DNA synthesis and repair. Although iron is necessary for class Ia RNR activity, little is known about the mechanisms that control RNR in response to iron deficiency. In this work, we demonstrate that yeast cells control RNR function during iron deficiency by redistributing the Rnr2-Rnr4 small subunit from the nucleus to the cytoplasm. Our data support a Mec1/Rad53-independent mechanism in which the iron-regulated Cth1/Cth2 mRNA-binding proteins specifically interact with the WTM1 mRNA in response to iron scarcity and promote its degradation. The resulting decrease in the nuclear-anchoring Wtm1 protein levels leads to the redistribution of the Rnr2-Rnr4 heterodimer to the cytoplasm, where it assembles as an active RNR complex and increases deoxyribonucleoside triphosphate levels. When iron is scarce, yeast selectively optimizes RNR function at the expense of other non-essential iron-dependent processes that are repressed, to allow DNA synthesis and repair.     

Respiration-linked proton translocation coupled to anaerobic reduction of manganese(IV) and iron(III) in Shewanella putrefaciens MR-1.

An oxidant pulse technique, with lactate as the electron donor, was used to study respiration-linked proton translocation in the manganese- and iron-reducing bacterium Shewanella putrefaciens MR-1. Cells grown anaerobically with fumarate or nitrate as the electron acceptor translocated protons in response to manganese (IV), fumarate, or oxygen. Cells grown anaerobically with fumarate also translocated protons in response to iron(III) and thiosulfate, whereas those grown with nitrate did not. Aerobically grown cells translocated protons only in response to oxygen. Proton translocation with all electron acceptors was abolished in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (20 microM) and was partially to completely inhibited by the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide (50 microM).     

Role of menaquinone in the reduction of fumarate, nitrate, iron(III) and manganese(IV) by Shewanella putrefaciens MR-1

Abstract Mutants of Shewanella putrefaciens MR-1 deficient in menaquinone and methylmenaquinone, but which have wild-type levels of ubiquinone, retain the ability to use trimethylamine N -oxide as an electron acceptor, but they lose the ability to use nitrate, iron(III), and fumarate as electron acceptors. These mutants also show a reduced rate of manganese(IV) reduction. One of these mutants could be restored to essentially wild-type phenotype by supplementing the medium with 1,4-dihydroxy-2-naphthoic acid. A requirement for naphthoquinones in iron(III) reduction and a preference for naphthoquinones in manganese(IV) reduction provide further support that the metal reducing systems in MR-1 are linked to anaerobic respiration.     

Nucleotide and thioredoxin specificity of the manganese ribonucleotide reductase from Brevibacterium ammoniagenes

The manganese-containing ribonucleotide reductase previously identified in gram-positive bacteria has been purified and its nucleotide specificity and other requirements were determined. The enzyme isolated from Brevibacterium ammoniagenes is a ribonucleoside-diphosphate reductase which, in the presence of allosteric effectors, reduces all four common substrates at comparable rates; very little activity is observed in the absence of effector nucleotides. Ribonucleoside triphosphates are reduced at 20% the rate of the diphosphates. Cytidine and uridine nucleotide reduction is specifically stimulated by ATP and dATP, adenylate reduction by dGTP, and guanosine nucleotide reduction by dTTP. Unlike the iron-containing ribonucleotide reductase systems, high concentrations of dATP do not inhibit substrate reduction. The new bacterial enzyme tolerates high salt concentrations (up to 250 mM ionic strength) and does not require divalent metal ions for activity in vitro. The presence of thioredoxin has been demonstrated in heat- and acid-treated protein extracts of B. ammoniagenes and the protein was purified to homogeneity. It is very similar to the thioredoxins isolated from other organisms in relative molecular mass (12,000), isoelectric point (4.3) and enzyme-activating properties. In the presence of 0.3 mM dithiothreitol, the bacterial thioredoxin can serve as hydrogen donor for B. ammoniagenes ribonucleotide reductase in vitro, indicating the presence of a functional ribonucleotide reductase-thioredoxin system in these bacteria. The properties described in this and in our preceding paper in this journal [Eur. J. Biochem. 170, 603-611 (1988)] suggest that the B. ammoniagenes ribonucleotide reductase is intermediate in structure and specificity between the deoxyadenosylcobalamin-dependent and the iron-containing enzyme classes and that it is adapted to the specific requirements of deoxyribonucleotide synthesis in this organism.     

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gr?slund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.     

Bacillus subtilis class Ib ribonucleotide reductase is a dimanganese(III)-tyrosyl radical enzyme

Bacillus subtilis class Ib ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, providing the building blocks for DNA replication and repair. It is composed of two proteins: α (NrdE) and β (NrdF). β contains the metallo-cofactor, essential for the initiation of the reduction process. The RNR genes are organized within the nrdI-nrdE-nrdF-ymaB operon. Each protein has been cloned, expressed, and purified from Escherichia coli. As isolated, recombinant NrdF (rNrdF) contained a diferric-tyrosyl radical [Fe(III)(2)-Y(?)] cofactor. Alternatively, this cluster could be self-assembled from apo-rNrdF, Fe(II), and O(2). Apo-rNrdF loaded using 4 Mn(II)/β(2), O(2), and reduced NrdI (a flavodoxin) can form a dimanganese(III)-Y(?) [Mn(III)(2)-Y(?)] cofactor. In the presence of rNrdE, ATP, and CDP, Mn(III)(2)-Y(?) and Fe(III)(2)-Y(?) rNrdF generate dCDP at rates of 132 and 10 nmol min(-1) mg(-1), respectively (both normalized for 1 Y(?)/β(2)). To determine the endogenous cofactor of NrdF in B. subtilis, the entire operon was placed behind a Pspank(hy) promoter and integrated into the B. subtilis genome at the amyE site. All four genes were induced in cells grown in Luria-Bertani medium, with levels of NrdE and NrdF elevated 35-fold relative to that of the wild-type strain. NrdE and NrdF were copurified in a 1:1 ratio from this engineered B. subtilis. The visible, EPR, and atomic absorption spectra of the purified NrdENrdF complex (eNrdF) exhibited characteristics of a Mn(III)(2)-Y(?) center with 2 Mn/β(2) and 0.5 Y(?)/β(2) and an activity of 318-363 nmol min(-1) mg(-1) (normalized for 1 Y(?)/β(2)). These data strongly suggest that the B. subtilis class Ib RNR is a Mn(III)(2)-Y(?) enzyme.     

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., J?rnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.     

Mechanism of assembly of the Bis(Molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase

A fully defined in vitro system has been developed for studying the mechanism of assembly of the bis(molybdopterin guanine dinucleotide)molybdenum cofactor in Rhodobacter sphaeroides dimethyl sulfoxide reductase (DMSOR). R. sphaeroides DMSOR expressed in a mobA(-) Escherichia coli strain lacks molybdopterin and molybdenum but contains a full complement of guanine in the form of GMP and GDP. Escherichia coli MobA, molybdopterin-Mo, GTP, and MgCl(2) are required and sufficient for the in vitro activation of purified DMSOR expressed in the absence of MobA. High levels of MobA inhibit the in vitro activation. A chaperone is not required for the in vitro activation process. The reconstituted DMSOR can exhibit up to 73% of the activity observed in recombinant DMSOR purified from a wild-type strain. The use of radiolabeled GTP has demonstrated incorporation of the guanine moiety from the GTP into the activated DMSOR. No role was observed for E. coli MobB in the in vitro activation of apo-DMSOR. This work also represents the first time that the MobA-mediated conversion of molybdopterin to molybdopterin guanine dinucleotide has been demonstrated directly without using the activation of a molybdoenzyme as an indicator for cofactor formation.     

Adducts of guanine with chromium(III), iron(III) and oxovanadium(IV) chlorides

The preparation of well-defined adducts of the M(guH)(₂Cl₃ (M = Cr, Fe) and VO(guH)Cl₂ types (guH = neutral guanine), by refluxing ligand and metal chloride mixtures in ethanol-triethyl orthoformate, is reported. Characterization studies suggest that the new complexes are probably linear chain-like polymeric species, involving single bridges of bidentate guH ligands between adjacent metal ions. Bidentate bridging guH is most probably coordinated through the N(7) and N(9) imidazole nitrogens. The chloro ligands present in the adducts are exclusively terminal. Infrared evidence rules out the possibility of coordination of guanine through either of its exocyclic potential binding sites (i.e., CO oxygen and NH₂ nitrogen) [1].     

Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone

AIMS: To clarify the inhibitory effects of ozone on Chlamydia trachomatis and C. pneumoniae. METHODS AND RESULTS: Cell culture was performed using HeLa229 cells for C. trachomatis, and Human Line cells for C. pneumoniae. C. trachomatis strain D/UW-3/Cx and C. pneumoniae strain AR-39 were used. Ozone water was generated by an ozone water dispenser and diluted to desired concentration just before each experiment. Preinoculation minimum cidal concentration (MCC) and postinoculation MCC methods were employed. In preinoculation MCC, chlamydial strains were treated with serially diluted ozone water followed by inoculation to cells. In postinoculation method, chlamydial strains were inoculated to cells and incubated for 24 h. Then infected cells were treated with ozone water, followed by additional incubation for 48 h. Complete inactivation was obtained in preinoculation MCC method at 0.5 ppm of ozone water for 30 s, or 4 ppm for 5 s. CONCLUSION: Ozone at a concentration of 4 ppm was enough for immediate inactivation of both C. trachomatis and C. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Ozone water at 4 ppm should be applicable for prevention of C. trachomatis urogenital infections.