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The class Ib ribonucleotide reductase of Escherichio coli can initiate reduction of nucleotides to deoxynucleotides with either a [Mn.sup.III.sub.2]-tyrosyl radical (Y*) or a [Fe.sup.III.sub.2]-Y* cofactor in the NrdF subunit. Whereas [Fe.sup.III.sub.2]-Y* can self-assemble from [Fe.sup.II.sub.2]-NrdF and [O.sub.2], activation of [Mn.sup.II.sub.2]-NrdF requires a reduced flavoprotein, Nrdl, proposed to form the oxidant for cofactor assembly by reduction of [O.sub.2]. The crystal structures reported here of E. coli [Mn.sup.II.sub.2]-NrdF and [Fe.sup.II.sub.2]-NrdF reveal different coordination environments, suggesting distinct initial binding sites for the oxidants during cofactor activation. In the structures of [Mn.sup.II.sub.2]-NrdF in complex with reduced and oxidized Nrdl, a continuous channel connects the Nrdl flavin cofactor to the NrdF [Mn.sup.II.sub.2] active site. Crystallographic detection of a putative peroxide in this channel supports the proposed mechanism of [Mn.sup.III.sub.2]-Y* cofactor assembly. 10.1126/science.1190187

Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo 'replication fork reversal.' This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

A study result which show that the class Ic ribonucleotide reductase from the human pathogen Chlamydia trachomatis which uses a [Mn.sup.IV]/[Fe.sup.III] cofactor to generate protein and substrate radicals in its catalytic mechanism is presented.

Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase intermediate X and three computationally derived model complexes are presented. Rapid freeze-quench (RFQ) EPR, absorption and MCD are used to trap intermediate X in R2 wild type and two variants.

Escherichia coli possesses class Ia, class Ib, and class III ribonucleotide reductases (RNR). Under standard laboratory conditions, the aerobic class Ia nrdAB RNR genes are well expressed, whereas the aerobic class Ib nrdEF RNR genes are poorly expressed. The class III RNR is normally expressed under microaerophilic and anaerobic conditions. In this paper, we show that the E. coli YbaD protein differentially regulates the expression of the three sets of genes. YbaD is a homolog of the Streptomyces NrdR protein. It is not essential for growth and has been renamed NrdR. Previously, Streptomyces NrdR was shown to transcriptionally regulate RNR genes by binding to specific 16-bp sequence motifs, NrdR boxes, located in the regulatoR' regions of its RNR operons. All three E. coli RNR operons contain two such NrdR box motifs positioned in their regulator regions. The NrdR boxes are located near to or overlap with the promoter elements. DNA binding experiments showed that NrdR binds to each of the upstream regulatory regions. We constructed deletions in nrdR (ybaD) and showed that they caused high-level induction of transcription of the class Ib RNR genes but had a much smaller effect on induction of transcription of the class Ia and class III RNR genes. We propose a model for differential regulation of the RNR genes based on binding of NrdR to the regulator' regions. The model assumes that differences in the positions of the NrdR binding sites, and in the sequences of the motifs themselves, determine the extent to which NrdR represses the transcription of each RNR operon.

Mobile genetic elements have the potential to influence the expression of genes surrounding their insertion site upon invasion of a genome. Here, we examine the transcriptional organization of a ribonucleotide reductase operon (nrd) that has been invaded by an HNH family homing endonuclease, mobE. In Aeromonas hydrophila phage Aehl, mobE has inserted into the large-subunit gene (nrdA) of aerobic ribonucleotide reductase (RNR), splitting it into two smaller genes, nrdA-a and nrdA-b. This gene organization differs from that in phages T4, T6, RB2, RB3, RB15, and LZ7, where mobE is inserted in the nrdA-nrdB intergenic region. We present evidence that the expression of Aehl mobE is regulated by transcriptional, posttranscriptional, and translational controls. An Aehl-specific late promoter drives expression of mobE, but strikingly the mobE transcript is processed internally at an RNase E-like site. We also identified a putative stem-loop structure upstream of mobE that sequesters the mobE ribosome binding site, presumably acting to down regulate MobE translation. Moreover, our transcriptional analyses indicate that the surrounding nrd genes of phage Aeh1 are expressed by a different strategy than are the corresponding phage T4 genes and that transcriptional readthrough is the only mechanism by which the promoterless Aeh1 nrdB gene is expressed. We suggest that the occurrence of multiple layers of control to limit the expression of mobE to late in the Aeh1 infection cycle is an adaptation of Aeh1 to reduce any effects on expression of the surrounding nrd genes early in phage infection when RNR function is critical.

Newly developed PCR assays were used to PCR-amplify and sequence fragments of the dissimilatory adenosine-5'-phosphosulfate (APS) reductase genes (aprBA) comprising nearly the entire gene locus (2.2-2-4 kb, equal to 92-94% of the protein coding sequence) from 75 sulfate-reducing prokaryotes (SRP) of a taxonomically wide range. Comparative phylogenetic analysis included all determined and publicly available AprBA sequences from SRP and selected homologous sequences of sulfur-oxidizing bacteria (SOB). The almost identical AprB and AprA tree topologies indicated a shared evolutionary path for the aprBA among the investigated SRP by vertical inheritance and concomitant lateral gene transfer (LGT). The topological comparison of AprB/A- and 16S rRNA gene-based phylogenetic trees revealed novel LGT events across the SRP divisions. Compositional gene analysis confirmed Thermacetogenium phaeum to be the first validated strain affected by a recent lateral transfer of aprBA as a putative effect of long-term co-cultivation with a Thermodesulfovibrio species. Interestingly, the Apr proteins of SRP and SOB diverged into two phylogenetic lineages, with the SRP affiliated with the green sulfur bacteria, e.g. Chlorobaculum tepidum, while the Aflochromatium vinosum-related sequences formed a distinct group. Analysis of genome data indicated that this phylogenetic separation is also reflected in the differing presence of the putative proteins functionally associated with Apr, QmoABC complex (quinone-interacting membrane-bound oxidoreductase) and AprM (transmembrane protein). Scenarios for the origin and evolution of the dissimilatory APS reductase are discussed within the context of the dissimilatory sulfite reductase (DsrAB) phylogeny, the appearance of QmoABC and AprM in the SRP and SOB genomes, and the geochemical setting of Archean Earth.

The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aerugiuosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aerugiuosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF long.

Tetrahydrofolate is a ubiquitous [C.sub.1] carrier in many biosynthetic pathways in bacteria, importantly, in the biosynthesis of formylmethionyl [tRNA.sup.fMET], which is essential for the initiation of translation. The final step in the biosynthesis of tetrahydrofolate is carried out by the enzyme dihydrofolate reductase (DHFR). A search of the complete genome sequence of Helicobacter pylori failed to reveal any sequence that encodes DHFR. Previous studies demonstrated that the H. pylori dihydropteroate synthase gene folP can complement an Escherichia coli strain in which folA and folM, encoding two distinct DHFRs, are deleted. It was also shown that H. pylori FolP possesses an additional N-terminal domain that binds flavin mononucleotide (FMN). Homologous domains are found in FolP proteins of other microorganisms that do not possess DHFR. In this study, we demonstrated that H. pylori FolP is also a dihydropteroate reductase that derives its reducing power from soluble flavins, reduced FMN and reduced flavin adenine dinucleotide. We also determined the stoichiometry of the enzymebound flavin and showed that half of the bound flavin is exchangeable with the soluble flavins. Finally, site-directed mutagenesis of the most conserved amino acid residues in the N-terminal domain indicated the importance of these residues for the activity of the enzyme as a dihydropteroate reductase.

The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a [Rec.sup.+] context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named 'replication fork reversal.' Viability results supported the occurrence of this process, as specific lethality was observed in the nrdA101 recB double mutant and was suppressed by the additional inactivation of ruvABC. None of these effects seem to be due to the limitation of the deoxyribonucleotide supply in the nrdA101 strain even at the permissive temperature, as we found the same level of DNA double-strand breaks in the [nrdA.sup.+] strain growing under limited (2-[micro]g/ml) or under optimal (5-[micro]g/ml) thymidine concentrations. We propose that the presence of an altered NDP reductase, as a component of the replication machinery, impairs the progression of the replication fork, contributing to the lengthening of the C period in the nrdA101 mutant at the permissive temperature. doi:10.1128/JB.01632-06

In bacterial and phage genomes, coding regions are sometimes interrupted by self-splicing introns or inteins, which can encode mobility-promoting homing endonucleases. Homing endonuclease genes are also found free-standing (not intron- or intein-encoded) in phage genomes where they are inserted in intergenic regions. One example is the HNH family endonuclease, mobE, inserted between the large (nrdA) and small (nrdB) subunit genes of aerobic ribonucleotide reductase (RNR) of T-even phages T4, RB2, RB3, RB15, and LZ7. Here, we describe an insertion of mobE into the nrdA gene of Aeromonas hydrophila phage Aeh1. The insertion creates a unique genes-in-pieces arrangement, where nrdA is split into two independent genes, nrdA-a and nrdA-b, each encoding cysteine residues that correspond to the active-site residues of uninterrupted NrdA proteins. Remarkably, the mobE insertion does not inactivate NrdA function, although the insertion is not a self-splicing intron or intein. We copurified the NrdA-a, NrdA-b, and NrdB proteins as complex from Aeh1-infected cells and also showed that a reconstituted complex has RNR activity. Class I RNR activity in phage Aeh1 is thus assembled from separate proteins that interact to form a composite active site, demonstrating that the mobE insertion is phenotypically neutral in that its presence as an intervening sequence does not disrupt the function of the surrounding gene. bacteriophage Aeh1 | gene structure | intervening sequence

A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.

In Chlamydiaceae, the nucleotide sequence between the 5S rRNA gene and the gene for subunit F of the N[a.sup.+]-translocating NADH-quinone reductase (nqrF or dmpP) has varied lengths and gene contents. We analyzed this site in 45 Chlamydiaceae strains having diverse geographical and pathological origins and including members of all nine species.

In the denitrifying bacterium Thauera aromatica, the central intermediate of anaerobic aromatic metabolism, benzoyl-coenzyme A (CoA), is dearomatized by the ATP-dependent benzoyl-CoA reductase to cyciohexa-l,5-diene-l-carbonyl-CoA (dienoyl-CoA). The dienoyl-CoA is further metabolized by a series of [beta]-oxidation-like reactions of the so-called benzoyl-CoA degradation pathway resulting in ring cleavage. Recently, evidence was obtained that obligately anaerobic bacteria that use aromatic growth substrates do not contain an ATP-dependent benzoyl-CoA reductase. In these bacteria, the reactions involved in dearomatization and cleavage of the aromatic ring have not been shown, so far. In this work, a characteristic enzymatic step of the benzoyl-CoA pathway in obligate anaerobes was demonstrated and characterized. Dienoyl-CoA hydratase activities were determined in extracts of Geobacter metallireducens (iron reducing), Syntrophus aciditrophicus (fermenting), and Desulfococcus multivorans (sulfate reducing) cells grown with benzoate. The benzoate-induced genes putatively coding for the dienoyl-CoA hydratases in the benzoate degraders G. metallireducens and S. aciditrophieus were heterologously expressed and characterized. Both gene products specifically catalyzed the reversible hydration of dienoyl-CoA to 6-hydroxycyclohexenoyl-CoA ([K.sub.m], 80 and 35 [micro]M; [V.sub.max], 350 and 550 [micro]mol [min.sup.-1] [mg.sup.-], respectively). Neither enzyme had significant activity with cyclohex-l-ene-l-carbonyl-CoA or crotonyl-CoA. The results suggest that benzoyl-CoA degradation proceeds via dienoyl-CoA and 6-hydroxycyclohexanoyl-CoA in strictly anaerobic bacteria. The steps involved in dienoyl-CoA metabolism appear identical in all nonphotosynthetic anaerobic bacteria, although totally different benzene ring-dearomatizing enzymes are present in facultative and obligate anaerobes.

In eukaryotic cells the concentration of dNTP is highest in S phase and lowest in [G.sub.1] phase and is controlled by ribonucleotide reductase (RNR). RNR activity is eliminated in all eukaryotes in [G.sub.1] phase by a variety of mechanisms: transcriptional regulation, small inhibitory proteins, and protein degradation. After activation of RNR upon commitment to S phase, dATP feedback inhibition ensures that the dNTP concentration does not exceed a certain maximal level. It is not apparent why limitation of dNTP concentration is necessary in [G.sub.1] phase. In principle, dATP feedback inhibition should be sufficient to couple dNTP production to utilization. We demonstrate that in Saccharomyces cerevisiae constitutively high dNTP concentration transiently arrests cell cycle progression in late [G.sub.1] phase, affects activation of origins of replication, and inhibits the DNA damage checkpoint. We propose that fluctuation of dNTP concentration controls cell cycle progression and the initiation of DNA replication. DNA replication | ribonucleotide reductase

Ribonucleotide reduetases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.

The Fe(IV) d-d transition energies for four active-site structural models of class I ribonucleotide reductase (RNR) intermediate X are calculated using broken-symmetry density functional theory incorporated with the Slater transition state vertical self-consistent reaction field methodology. This model containing two mu-oxo bridges, one terminal water, and one bidentate carboxylate group, was found to yield the best Fe(IV) d-d transition energies compared with experiment.

The studies have shown that the mechanism is one of the two that are operant in hydroxyurea (HU) inactivation of the Chlamydia trachomatis (Ct) enzyme. The combined requirements for the catalytic subunit, the substrate and a functional surface-to-cofactor electron relay system has shown that HU effects the [Mn.sup.IV]/[Fe.sup.III] -> [Mn.sup.III]/[Fe.sup.III] reduction by intercepting a stable tyrosyl radical that forms when the ready holoenzyme complex is assembled, the electron transfer gate is opened, and the [Mn.sup.IV] oxidizes either Y222 or Y338.

Photochemical ribonucleotide reductases (photoRNRs) were developed to study the proton-coupled electron transfer (PCET) mechanism of radical transport in Escherichia coli class I ribonucleotide reductase (RNR). The biexponential kinetics observed for PCET suggested a large degree of conformational flexibility in the [Re]-Y-[Re]-Y-[beta]C19- [alpha]2 complex that engendered partitioning of the N-terminus of the peptide into both bound and solvent-exposed fractions.

A new method was developed to determine the molecular basis for interactions between the subunits of ribonucleotide reductase (RNR) and to measure their affinities in the presence of different substrate and effector pairs. The developed method could also be applied to RNRs from other sources and gain insight into the complexities of other flexible proteins.

The proton-coupled electron transfer (PCET) of radical transport in ribonucleotide reductase (RNR) is studied by using engineered photoRNRs. The [Re] chromophore with fluorotyrosines has provided a method for selectively generating tyrosyl radicals on the [F.sub.R]Y-R2C19 peptide bound to [alpha]2 that are competent for turnover and that can be seen with transient absorption (TA) spectroscopy.

The kinetics of decay of the W[48.sup.+.] in the absence of reductants in the reactions of the Escherichia coli wild-type R2 protein and its Y122F, Y356F, and Y122F/Y356 variants were characterized. It was seen that the presence of [Mg.sup.2+] at concentration similar to that used in ribonucleotide reductase activity assays brings Y356 into a rapid radical-transfer equilibrium with the W[48.sup.+.] leading to a very rapid phase of Y. formation (Y356.) and an efficient pathway for decay of the W[48.sup.+.].

Ribonucleotide reductases (RNRs) catalyze the synthesis of the four-deoxyribonucleoside triphosphates (dNTPs) required for DNA replication and repair by the substitution of the 2'OH-group of a ribonucleoside di- or triphosphate with a hydrogen atom using a mechanism involving protein radicals. An intricate interplay between gene activation, enzyme inhibition, and protein degradation regulates, along with the allosteric effects, the activity of RNR and provides the appropriate amount of dNTPs for DNA replication and repair.