Identification of the TyrOH•+ Radical Cation in the Flavoenzyme TrmFO.

Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOH•+, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOH•+ is formed in ∼1 ps by electron transfer to excited flavin and decays in ∼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates.

Targeting of Helicobacter pylori thymidylate synthase ThyX by non-mitotoxic hydroxy-naphthoquinones.

ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.

Hotspots in an obligate homodimeric anticancer target. Structural and functional effects of interfacial mutations in human thymidylate synthase.

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.

Transcriptional activation and cell cycle block are the keys for 5-fluorouracil induced up-regulation of human thymidylate synthase expression.

BACKGROUND: 5-fluorouracil, a commonly used chemotherapeutic agent, up-regulates expression of human thymidylate synthase (hTS). Several different regulatory mechanisms have been proposed to mediate this up-regulation in distinct cell lines, but their specific contributions in a single cell line have not been investigated to date. We have established the relative contributions of these previously proposed regulatory mechanisms in the ovarian cancer cell line 2008 and the corresponding cisplatin-resistant and 5-FU cross-resistant-subline C13*. METHODOLOGY/PRINCIPAL FINDINGS: Using RNA polymerase II inhibitor DRB treated cell cultures, we showed that 70-80% of up-regulation of hTS results from transcriptional activation of TYMS mRNA. Moreover, we report that 5-FU compromises the cell cycle by blocking the 2008 and C13* cell lines in the S phase. As previous work has established that TYMS mRNA is synthesized in the S and G(1) phase and hTS is localized in the nuclei during S and G(2)-M phase, the observed cell cycle changes are also expected to affect the intracellular regulation of hTS. Our data also suggest that the inhibition of the catalytic activity of hTS and the up-regulation of the hTS protein level are not causally linked, as the inactivated ternary complex, formed by hTS, deoxyuridine monophosphate and methylenetetrahydrofolate, was detected already 3 hours after 5-FU exposure, whereas substantial increase in global TS levels was detected only after 24 hours. CONCLUSIONS/SIGNIFICANCE: Altogether, our data indicate that constitutive TYMS mRNA transcription, cell cycle-induced hTS regulation and hTS enzyme stability are the three key mechanisms responsible for 5-fluorouracil induced up-regulation of human thymidylate synthase expression in the two ovarian cancer cell lines studied. As these three independent regulatory phenomena occur in a precise order, our work provides a feasible rationale for earlier observed synergistic combinations of 5-FU with other drugs and may suggest novel therapeutic strategies.

Insights into folate/FAD-dependent tRNA methyltransferase mechanism: role of two highly conserved cysteines in catalysis.

The flavoprotein TrmFO methylates specifically the C5 carbon of the highly conserved uridine 54 in tRNAs. Contrary to most methyltransferases, the 1-carbon unit transferred by TrmFO derives from 5,10-methylenetetrahydrofolate and not from S-adenosyl-L-methionine. The enzyme also employs the FAD hydroquinone as a reducing agent of the C5 methylene U54-tRNA intermediate in vitro. By analogy with the catalytic mechanism of thymidylate synthase ThyA, a conserved cysteine located near the FAD isoalloxazine ring was proposed to act as a nucleophile during catalysis. Here, we mutated this residue (Cys-53 in Bacillus subtilis TrmFO) to alanine and investigated its functional role. Biophysical characterization of this variant demonstrated the major structural role of Cys-53 in maintaining both the integrity and plasticity of the flavin binding site. Unexpectedly, gel mobility shift assays showed that, like the wild-type enzyme, the inactive C53A variant was capable of forming a covalent complex with a 5-fluorouridine-containing mini-RNA. This result confirms the existence of a covalent intermediate during catalysis but rules out a nucleophilic role for Cys-53. To identify the actual nucleophile, two other strictly conserved cysteines (Cys-192 and Cys-226) that are relatively far from the active site were replaced with alanine, and a double mutant C53A/C226A was generated. Interestingly, only mutations that target Cys-226 impeded TrmFO from forming a covalent complex and methylating tRNA. Altogether, we propose a revised mechanism for the m(5)U54 modification catalyzed by TrmFO, where Cys-226 attacks the C6 atom of the uridine, and Cys-53 plays the role of the general base abstracting the C5 proton.

The structure of an archaeal homodimeric ligase which has RNA circularization activity.

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 A of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.

A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen.

During DNA replication and repair, many proteins bind to and dissociate in a highly specific and ordered manner from proliferating cell nuclear antigen (PCNA). We describe a combined approach of in silico searches at the genome level and combinatorial peptide synthesis to investigate the binding properties of hundreds of short PCNA-interacting peptides (PIP-peptides) to archaeal and eukaryal PCNAs. Biological relevance of our combined approach was demonstrated by identification an inactive complex of Pyrococcus abyssi ribonuclease HII with PCNA. Furthermore we show that PIP-peptides interact with PCNA largely in a sequence independent manner. Our experimental approach also identified many so far unidentified PCNA interacting peptides in a number of human proteins.

In vitro detection of the enzymatic activity of folate-dependent tRNA (Uracil-54,-C5)-methyltransferase: evolutionary implications.

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA[uracil-54,C5]-MTases). In eukaryotes and many bacteria, the methyl donor for this reaction is generally S-adenosyl-L-methionine (S-AdoMet). However, in other bacteria, like Enterococcus faecalis and Bacillus subtilis, it was shown that the source of carbon is N(5),N(10)-methylenetetrahydrofolate (CH(2)=THF). Recently we have determined that the Bacillus subtilis gid gene (later renamed to trmFO) encodes the folate-dependent tRNA(uracil-54,C5)-MTase. Here, we describe a procedure for overexpression and purification of this recombinant enzyme, as well as detection of its activity in vitro. Inspection of presently available sequenced genomes reveals that trmFO gene is present in most Firmicutes, in all alpha- and delta-Proteobacteria (except Rickettsiales in which the trmFO gene is missing), Deinococci, Cyanobacteria, Fusobacteria, Thermotogales, Acidobacteria, and in one Actinobacterium. Interestingly, trmFO is never found in genomes containing the gene trmA coding for S-adenosyl-L-methionine-dependent tRNA (uracil-54,C5)-MTase. The phylogenetic analysis of TrmFO sequences suggests an ancient origin of this enzyme in bacteria.

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp.

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.

Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications.

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m(5)U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N(5), N(10)-methylenetetrahydrofolate (CH(2)H(4)folate). We have identified the gene for Bacillus subtilis tRNA:m(5)U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m(5)U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor.

Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation.

The mechanisms by which hyperthermophilic Archaea, such as "Pyrococcus abyssi" and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of "P. abyssi" chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated "P. abyssi" cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated "P. abyssi" cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that "P. abyssi" contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.

Identification, purification, and characterization of an eukaryotic-like phosphopantetheine adenylyltransferase (coenzyme A biosynthetic pathway) in the hyperthermophilic archaeon Pyrococcus abyssi.

Although coenzymeA (CoA) is essential in numerous metabolic pathways in all living cells, molecular characterization of the CoA biosynthetic pathway in Archaea remains undocumented. Archaeal genomes contain detectable homologues for only three of the five steps of the CoA biosynthetic pathway characterized in Eukarya and Bacteria. In case of phosphopantetheine adenylyltransferase (PPAT) (EC 2.7.7.3), the putative archaeal enzyme exhibits significant sequence similarity only with its eukaryotic homologs, an unusual situation for a protein involved in a central metabolic pathway. We have overexpressed in Escherichia coli, purified, and characterized this putative PPAT from the hyperthermophilic archaeon Pyrococcus abyssi (PAB0944). Matrix-assisted laser desorption ionization-time of flight mass spectrometry and high performance liquid chromatography measurements are consistent with the presence of a dephospho-CoA (dPCoA) molecule tightly bound to the polypeptide. The protein indeed catalyzes the synthesis of dPCoA from 4'-phosphopantetheine and ATP, as well as the reverse reaction. The presence of dPCoA stabilizes PAB0944, as it induces a shift from 76 to 82 degrees C of the apparent Tm measured by differential scanning microcalorimetry. Potassium glutamate was found to stabilize the protein at 400 mm. The enzyme behaves as a monomeric protein. Although only distantly related, secondary structure prediction indicates that archaeal and eukaryal PPAT belong to the same nucleotidyltransferase superfamily of bacterial PPAT. The existence of operational proteins highly conserved between Archaea and Eukarya involved in a central metabolic pathway challenge evolutionary scenarios in which eukaryal operational proteins are strictly of bacterial origin.