Exploring a unique class of flavoenzymes: Identification and biochemical characterization of ribosomal RNA dihydrouridine synthase.

Dihydrouridine (D), a prevalent and evolutionarily conserved base in the transcriptome, primarily resides in tRNAs and, to a lesser extent, in mRNAs. Notably, this modification is found at position 2449 in the Escherichia coli 23S rRNA, strategically positioned near the ribosome's peptidyl transferase site. Despite the prior identification, in E. coli genome, of three dihydrouridine synthases (DUS), a set of NADPH and FMN-dependent enzymes known for introducing D in tRNAs and mRNAs, characterization of the enzyme responsible for D2449 deposition has remained elusive. This study introduces a rapid method for detecting D in rRNA, involving reverse transcriptase-blockage at the rhodamine-labeled D2449 site, followed by PCR amplification (RhoRT-PCR). Through analysis of rRNA from diverse E. coli strains, harboring chromosomal or single-gene deletions, we pinpoint the yhiN gene as the ribosomal dihydrouridine synthase, now designated as RdsA. Biochemical characterizations uncovered RdsA as a unique class of flavoenzymes, dependent on FAD and NADH, with a complex structural topology. In vitro assays demonstrated that RdsA dihydrouridylates a short rRNA transcript mimicking the local structure of the peptidyl transferase site. This suggests an early introduction of this modification before ribosome assembly. Phylogenetic studies unveiled the widespread distribution of the yhiN gene in the bacterial kingdom, emphasizing the conservation of rRNA dihydrouridylation. In a broader context, these findings underscore nature's preference for utilizing reduced flavin in the reduction of uridines and their derivatives.

Functional redundancy in tRNA dihydrouridylation.

Dihydrouridine (D) is a common modified base found predominantly in transfer RNA (tRNA). Despite its prevalence, the mechanisms underlying dihydrouridine biosynthesis, particularly in prokaryotes, have remained elusive. Here, we conducted a comprehensive investigation into D biosynthesis in Bacillus subtilis through a combination of genetic, biochemical, and epitranscriptomic approaches. Our findings reveal that B. subtilis relies on two FMN-dependent Dus-like flavoprotein homologs, namely DusB1 and DusB2, to introduce all D residues into its tRNAs. Notably, DusB1 exhibits multisite enzyme activity, enabling D formation at positions 17, 20, 20a and 47, while DusB2 specifically catalyzes D biosynthesis at positions 20 and 20a, showcasing a functional redundancy among modification enzymes. Extensive tRNA-wide D-mapping demonstrates that this functional redundancy impacts the majority of tRNAs, with DusB2 displaying a higher dihydrouridylation efficiency compared to DusB1. Interestingly, we found that BsDusB2 can function like a BsDusB1 when overexpressed in vivo and under increasing enzyme concentration in vitro. Furthermore, we establish the importance of the D modification for B. subtilis growth at suboptimal temperatures. Our study expands the understanding of D modifications in prokaryotes, highlighting the significance of functional redundancy in this process and its impact on bacterial growth and adaptation.

Structural basis of Qng1-mediated salvage of the micronutrient queuine from queuosine-5'-monophosphate as the biological substrate.

Eukaryotic life benefits from-and ofttimes critically relies upon-the de novo biosynthesis and supply of vitamins and micronutrients from bacteria. The micronutrient queuosine (Q), derived from diet and/or the gut microbiome, is used as a source of the nucleobase queuine, which once incorporated into the anticodon of tRNA contributes to translational efficiency and accuracy. Here, we report high-resolution, substrate-bound crystal structures of the Sphaerobacter thermophilus queuine salvage protein Qng1 (formerly DUF2419) and of its human ortholog QNG1 (C9orf64), which together with biochemical and genetic evidence demonstrate its function as the hydrolase releasing queuine from queuosine-5'-monophosphate as the biological substrate. We also show that QNG1 is highly expressed in the liver, with implications for Q salvage and recycling. The essential role of this family of hydrolases in supplying queuine in eukaryotes places it at the nexus of numerous (patho)physiological processes associated with queuine deficiency, including altered metabolism, proliferation, differentiation and cancer progression.

7-Deazaguanines in DNA: functional and structural elucidation of a DNA modification system.

The modified nucleosides 2'-deoxy-7-cyano- and 2'-deoxy-7-amido-7-deazaguanosine (dPreQ0 and dADG, respectively) recently discovered in DNA are the products of the bacterial queuosine tRNA modification pathway and the dpd gene cluster, the latter of which encodes proteins that comprise the elaborate Dpd restriction-modification system present in diverse bacteria. Recent genetic studies implicated the dpdA, dpdB and dpdC genes as encoding proteins necessary for DNA modification, with dpdD-dpdK contributing to the restriction phenotype. Here we report the in vitro reconstitution of the Dpd modification machinery from Salmonella enterica serovar Montevideo, the elucidation of the roles of each protein and the X-ray crystal structure of DpdA supported by small-angle X-ray scattering analysis of DpdA and DpdB, the former bound to DNA. While the homology of DpdA with the tRNA-dependent tRNA-guanine transglycosylase enzymes (TGT) in the queuosine pathway suggested a similar transglycosylase activity responsible for the exchange of a guanine base in the DNA for 7-cyano-7-deazaguanine (preQ0), we demonstrate an unexpected ATPase activity in DpdB necessary for insertion of preQ0 into DNA, and identify several catalytically essential active site residues in DpdA involved in the transglycosylation reaction. Further, we identify a modification site for DpdA activity and demonstrate that DpdC functions independently of DpdA/B in converting preQ0-modified DNA to ADG-modified DNA.

Four additional natural 7-deazaguanine derivatives in phages and how to make them.

Bacteriophages and bacteria are engaged in a constant arms race, continually evolving new molecular tools to survive one another. To protect their genomic DNA from restriction enzymes, the most common bacterial defence systems, double-stranded DNA phages have evolved complex modifications that affect all four bases. This study focuses on modifications at position 7 of guanines. Eight derivatives of 7-deazaguanines were identified, including four previously unknown ones: 2'-deoxy-7-(methylamino)methyl-7-deazaguanine (mdPreQ1), 2'-deoxy-7-(formylamino)methyl-7-deazaguanine (fdPreQ1), 2'-deoxy-7-deazaguanine (dDG) and 2'-deoxy-7-carboxy-7-deazaguanine (dCDG). These modifications are inserted in DNA by a guanine transglycosylase named DpdA. Three subfamilies of DpdA had been previously characterized: bDpdA, DpdA1, and DpdA2. Two additional subfamilies were identified in this work: DpdA3, which allows for complete replacement of the guanines, and DpdA4, which is specific to archaeal viruses. Transglycosylases have now been identified in all phages and viruses carrying 7-deazaguanine modifications, indicating that the insertion of these modifications is a post-replication event. Three enzymes were predicted to be involved in the biosynthesis of these newly identified DNA modifications: 7-carboxy-7-deazaguanine decarboxylase (DpdL), dPreQ1 formyltransferase (DpdN) and dPreQ1 methyltransferase (DpdM), which was experimentally validated and harbors a unique fold not previously observed for nucleic acid methylases.

Bacterial RNA Degradosomes: Molecular Machines under Tight Control.

Bacterial RNA degradosomes are multienzyme molecular machines that act as hubs for post-transcriptional regulation of gene expression. The ribonuclease activities of these complexes require tight regulation, as they are usually essential for cell survival while potentially destructive. Recent studies have unveiled a wide variety of regulatory mechanisms including autoregulation, post-translational modifications, and protein compartmentalization. Recently, the subcellular organization of bacterial RNA degradosomes was found to present similarities with eukaryotic messenger ribonucleoprotein (mRNP) granules, membraneless compartments that are also involved in mRNA and protein storage and/or mRNA degradation. In this review, we present the current knowledge on the composition and targets of RNA degradosomes, the most recent developments regarding the regulation of these machineries, and their similarities with the eukaryotic mRNP granules.

Alternate routes to mnm5s2U synthesis in Gram-positive bacteria.

The wobble bases of tRNAs that decode split codons are often heavily modified. In bacteria, tRNAGlu, Gln, Asp contains a variety of xnm5s2U derivatives. The synthesis pathway for these modifications is complex and fully elucidated only in a handful of organisms, including the Gram-negative Escherichia coli K12 model. Despite the ubiquitous presence of mnm5s2U modification, genomic analysis shows the absence of mnmC orthologous genes, suggesting the occurrence of alternate biosynthetic schemes for the conversion of cmnm5s2U to mnm5s2U. Using a combination of comparative genomics and genetic studies, a member of the YtqA subgroup of the radical Sam superfamily was found to be involved in the synthesis of mnm5s2U in both Bacillus subtilis and Streptococcus mutans. This protein, renamed MnmL, is encoded in an operon with the recently discovered MnmM methylase involved in the methylation of the pathway intermediate nm5s2U into mnm5s2U in B. subtilis. Analysis of tRNA modifications of both S. mutans and Streptococcus pneumoniae shows that growth conditions and genetic backgrounds influence the ratios of pathway intermediates owing to regulatory loops that are not yet understood. The MnmLM pathway is widespread along the bacterial tree, with some phyla, such as Bacilli, relying exclusively on these two enzymes. Although mechanistic details of these newly discovered components are not fully resolved, the occurrence of fusion proteins, alternate arrangements of biosynthetic components, and loss of biosynthetic branches provide examples of biosynthetic diversity to retain a conserved tRNA modification in Nature.IMPORTANCEThe xnm5s2U modifications found in several tRNAs at the wobble base position are widespread in bacteria where they have an important role in decoding efficiency and accuracy. This work identifies a novel enzyme (MnmL) that is a member of a subgroup of the very versatile radical SAM superfamily and is involved in the synthesis of mnm5s2U in several Gram-positive bacteria, including human pathogens. This is another novel example of a non-orthologous displacement in the field of tRNA modification synthesis, showing how different solutions evolve to retain U34 tRNA modifications.

Queuosine salvage in Bartonella henselae Houston 1: a unique evolutionary path.

Queuosine (Q) stands out as the sole tRNA modification that can be synthesized via salvage pathways. Comparative genomic analyses identified specific bacteria that showed a discrepancy between the projected Q salvage route and the predicted substrate specificities of the two identified salvage proteins: (1) the distinctive enzyme tRNA guanine-34 transglycosylase (bacterial TGT, or bTGT), responsible for inserting precursor bases into target tRNAs; and (2) queuosine precursor transporter (QPTR), a transporter protein that imports Q precursors. Organisms such as the facultative intracellular pathogen Bartonella henselae, which possess only bTGT and QPTR but lack predicted enzymes for converting preQ1 to Q, would be expected to salvage the queuine (q) base, mirroring the scenario for the obligate intracellular pathogen Chlamydia trachomatis. However, sequence analyses indicate that the substrate-specificity residues of their bTGTs resemble those of enzymes inserting preQ1 rather than q. Intriguingly, MS analyses of tRNA modification profiles in B. henselae reveal trace amounts of preQ1, previously not observed in a natural context. Complementation analysis demonstrates that B. henselae bTGT and QPTR not only utilize preQ1, akin to their Escherichia coli counterparts, but can also process q when provided at elevated concentrations. The experimental and phylogenomic analyses suggest that the Q pathway in B. henselae could represent an evolutionary transition among intracellular pathogens - from ancestors that synthesized Q de novo to a state prioritizing the salvage of q. Another possibility that will require further investigations is that the insertion of preQ1 confers fitness advantages when B. henselae is growing outside a mammalian host.

Integrative Approach to Probe Alternative Redox Mechanisms in RNA Modifications.

ConspectusRNA modifications found in most RNAs, particularly in tRNAs and rRNAs, reveal an abundance of chemical alterations of nucleotides. Over 150 distinct RNA modifications are known, emphasizing a remarkable diversity of chemical moieties in RNA molecules. These modifications play pivotal roles in RNA maturation, structural integrity, and the fidelity and efficiency of translation processes. The catalysts responsible for these modifications are RNA-modifying enzymes that use a striking array of chemistries to directly influence the chemical landscape of RNA. This diversity is further underscored by instances where the same modification is introduced by distinct enzymes that use unique catalytic mechanisms and cofactors across different domains of life. This phenomenon of convergent evolution highlights the biological importance of RNA modification and the vast potential within the chemical repertoire for nucleotide alteration. While shared RNA modifications can hint at conserved enzymatic pathways, a major bottleneck is to identify alternative routes within species that possess a modified RNA but are devoid of known RNA-modifying enzymes. To address this challenge, a combination of bioinformatic and experimental strategies proves invaluable in pinpointing new genes responsible for RNA modifications. This integrative approach not only unveils new chemical insights but also serves as a wellspring of inspiration for biocatalytic applications and drug design. In this Account, we present how comparative genomics and genome mining, combined with biomimetic synthetic chemistry, biochemistry, and anaerobic crystallography, can be judiciously implemented to address unprecedented and alternative chemical mechanisms in the world of RNA modification. We illustrate these integrative methodologies through the study of tRNA and rRNA modifications, dihydrouridine, 5-methyluridine, queuosine, 8-methyladenosine, 5-carboxymethylamino-methyluridine, or 5-taurinomethyluridine, each dependent on a diverse array of redox chemistries, often involving organic compounds, organometallic complexes, and metal coenzymes. We explore how vast genome and tRNA databases empower comparative genomic analyses and enable the identification of novel genes that govern RNA modification. Subsequently, we describe how the isolation of a stable reaction intermediate can guide the synthesis of a biomimetic to unveil new enzymatic pathways. We then discuss the usefulness of a biochemical "shunt" strategy to study catalytic mechanisms and to directly visualize reactive intermediates bound within active sites. While we primarily focus on various RNA-modifying enzymes studied in our laboratory, with a particular emphasis on the discovery of a SAM-independent methylation mechanism, the strategies and rationale presented herein are broadly applicable for the identification of new enzymes and the elucidation of their intricate chemistries. This Account offers a comprehensive glimpse into the evolving landscape of RNA modification research and highlights the pivotal role of integrated approaches to identify novel enzymatic pathways.

4'-Deoxypyridoxine disrupts vitamin B6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B6 uptake and PLP-dependent enzyme activity.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

Specificity in the biosynthesis of the universal tRNA nucleoside N6-threonylcarbamoyl adenosine (t6A)-TsaD is the gatekeeper.

N6-threonylcarbamoyl adenosine (t6A) is a nucleoside modification found in all kingdoms of life at position 37 of tRNAs decoding ANN codons, which functions in part to restrict translation initiation to AUG and suppress frameshifting at tandem ANN codons. In Bacteria the proteins TsaB, TsaC (or C2), TsaD, and TsaE, comprise the biosynthetic apparatus responsible for t6A formation. TsaC(C2) and TsaD harbor the relevant active sites, with TsaC(C2) catalyzing the formation of the intermediate threonylcarbamoyladenosine monophosphate (TC-AMP) from ATP, threonine, and CO2, and TsaD catalyzing the transfer of the threonylcarbamoyl moiety from TC-AMP to A37 of substrate tRNAs. Several related modified nucleosides, including hydroxynorvalylcarbamoyl adenosine (hn6A), have been identified in select organisms, but nothing is known about their biosynthesis. To better understand the mechanism and structural constraints on t6A formation, and to determine if related modified nucleosides are formed via parallel biosynthetic pathways or the t6A pathway, we carried out biochemical and biophysical investigations of the t6A systems from E. coli and T. maritima to address these questions. Using kinetic assays of TsaC(C2), tRNA modification assays, and NMR, our data demonstrate that TsaC(C2) exhibit relaxed substrate specificity, producing a variety of TC-AMP analogs that can differ in both the identity of the amino acid and nucleotide component, whereas TsaD displays more stringent specificity, but efficiently produces hn6A in E. coli and T. maritima tRNA. Thus, in organisms that contain modifications such as hn6A in their tRNA, we conclude that their origin is due to formation via the t6A pathway.

Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

Discovery of novel bacterial queuine salvage enzymes and pathways in human pathogens.

Queuosine (Q) is a complex tRNA modification widespread in eukaryotes and bacteria that contributes to the efficiency and accuracy of protein synthesis. Eukaryotes are not capable of Q synthesis and rely on salvage of the queuine base (q) as a Q precursor. While many bacteria are capable of Q de novo synthesis, salvage of the prokaryotic Q precursors preQ0 and preQ1 also occurs. With the exception of Escherichia coli YhhQ, shown to transport preQ0 and preQ1, the enzymes and transporters involved in Q salvage and recycling have not been well described. We discovered and characterized 2 Q salvage pathways present in many pathogenic and commensal bacteria. The first, found in the intracellular pathogen Chlamydia trachomatis, uses YhhQ and tRNA guanine transglycosylase (TGT) homologs that have changed substrate specificities to directly salvage q, mimicking the eukaryotic pathway. The second, found in bacteria from the gut flora such as Clostridioides difficile, salvages preQ1 from q through an unprecedented reaction catalyzed by a newly defined subgroup of the radical-SAM enzyme family. The source of q can be external through transport by members of the energy-coupling factor (ECF) family or internal through hydrolysis of Q by a dedicated nucleosidase. This work reinforces the concept that hosts and members of their associated microbiota compete for the salvage of Q precursors micronutrients.

Epoxyqueuosine Reductase QueH in the Biosynthetic Pathway to tRNA Queuosine Is a Unique Metalloenzyme.

Queuosine is a structurally unique and functionally important tRNA modification, widely distributed in eukaryotes and bacteria. The final step of queuosine biosynthesis is the reduction/deoxygenation of epoxyqueuosine to form the cyclopentene motif of the nucleobase. The chemistry is performed by the structurally and functionally characterized cobalamin-dependent QueG. However, the queG gene is absent from several bacteria that otherwise retain queuosine biosynthesis machinery. Members of the IPR003828 family (previously known as DUF208) have been recently identified as nonorthologous replacements of QueG, and this family was renamed QueH. Here, we present the structural characterization of QueH from Thermotoga maritima. The structure reveals an unusual active site architecture with a [4Fe-4S] metallocluster along with an adjacent coordinated iron metal. The juxtaposition of the cofactor and coordinated metal ion predicts a unique mechanism for a two-electron reduction/deoxygenation of epoxyqueuosine. To support the structural characterization, in vitro biochemical and genomic analyses are presented. Overall, this work reveals new diversity in the chemistry of iron/sulfur-dependent enzymes and novel insight into the last step of this widely conserved tRNA modification.

The DUF328 family member YaaA is a DNA-binding protein with a novel fold.

DUF328 family proteins are present in many prokaryotes; however, their molecular activities are unknown. The Escherichia coli DUF328 protein YaaA is a member of the OxyR regulon and is protective against oxidative stress. Because uncharacterized proteins involved in prokaryotic oxidative stress response are rare, we sought to learn more about the DUF328 family. Using comparative genomics, we found a robust association between the DUF328 family and genes involved in DNA recombination and the oxidative stress response. In some proteins, DUF328 domains are fused to other domains involved in DNA binding, recombination, and repair. Cofitness analysis indicates that DUF328 family genes associate with recombination-mediated DNA repair pathways, particularly the RecFOR pathway. Purified recombinant YaaA binds to dsDNA, duplex DNA containing bubbles of unpaired nucleotides, and Holliday junction constructs in vitro with dissociation equilibrium constants of 200-300 nm YaaA binds DNA with positive cooperativity, forming multiple shifted species in electrophoretic mobility shift assays. The 1.65-Å resolution X-ray crystal structure of YaaA reveals that the protein possesses a new fold that we name the cantaloupe fold. YaaA has a positively charged cleft and a helix-hairpin-helix DNA-binding motif found in other DNA repair enzymes. Our results demonstrate that YaaA is a new type of DNA-binding protein associated with the oxidative stress response and that this molecular function is likely conserved in other DUF328 family members.

Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes.

Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in Escherichia coli. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique dus gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using in vitro activity tests as well as in vivo E. coli complementation assays with the enzyme from Mycoplasma capricolum (DusBMCap), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusBMCap modifies U17 to D17 but also synthetizes D20/D20a combining therefore both E. coli DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the M. capricolum DusB enzyme could be an ancestral property.

Matching tRNA modifications in humans to their known and predicted enzymes.

tRNA are post-transcriptionally modified by chemical modifications that affect all aspects of tRNA biology. An increasing number of mutations underlying human genetic diseases map to genes encoding for tRNA modification enzymes. However, our knowledge on human tRNA-modification genes remains fragmentary and the most comprehensive RNA modification database currently contains information on approximately 20% of human cytosolic tRNAs, primarily based on biochemical studies. Recent high-throughput methods such as DM-tRNA-seq now allow annotation of a majority of tRNAs for six specific base modifications. Furthermore, we identified large gaps in knowledge when we predicted all cytosolic and mitochondrial human tRNA modification genes. Only 48% of the candidate cytosolic tRNA modification enzymes have been experimentally validated in mammals (either directly or in a heterologous system). Approximately 23% of the modification genes (cytosolic and mitochondrial combined) remain unknown. We discuss these 'unidentified enzymes' cases in detail and propose candidates whenever possible. Finally, tissue-specific expression analysis shows that modification genes are highly expressed in proliferative tissues like testis and transformed cells, but scarcely in differentiated tissues, with the exception of the cerebellum. Our work provides a comprehensive up to date compilation of human tRNA modifications and their enzymes that can be used as a resource for further studies.

An Unusual Route for p-Aminobenzoate Biosynthesis in Chlamydia trachomatis Involves a Probable Self-Sacrificing Diiron Oxygenase.

Chlamydia trachomatis lacks the canonical genes required for the biosynthesis of p-aminobenzoate (pABA), a component of essential folate cofactors. Previous studies revealed a single gene from C. trachomatis, the CT610 gene, that rescues Escherichia coli ΔpabA, ΔpabB, and ΔpabC mutants, which are otherwise auxotrophic for pABA. CT610 shares low sequence similarity to nonheme diiron oxygenases, and the previously solved crystal structure revealed a diiron active site. Genetic studies ruled out several potential substrates for CT610-dependent pABA biosynthesis, including chorismate and other shikimate pathway intermediates, leaving the actual precursor(s) unknown. Here, we supplied isotopically labeled potential precursors to E. coli ΔpabA cells expressing CT610 and found that the aromatic portion of tyrosine was highly incorporated into pABA, indicating that tyrosine is a precursor for CT610-dependent pABA biosynthesis. Additionally, in vitro enzymatic experiments revealed that purified CT610 exhibits low pABA synthesis activity under aerobic conditions in the absence of tyrosine or other potential substrates, where only the addition of a reducing agent such as dithiothreitol appears to stimulate pABA production. Furthermore, site-directed mutagenesis studies revealed that two conserved active site tyrosine residues are essential for the pABA synthesis reaction in vitro Thus, the current data are most consistent with CT610 being a unique self-sacrificing enzyme that utilizes its own active site tyrosine residue(s) for pABA biosynthesis in a reaction that requires O2 and a reduced diiron cofactor.IMPORTANCEChlamydia trachomatis is the most reported sexually transmitted infection in the United States and the leading cause of infectious blindness worldwide. Unlike many other intracellular pathogens that have undergone reductive evolution, C. trachomatis is capable of de novo biosynthesis of the essential cofactor tetrahydrofolate using a noncanonical pathway. Here, we identify the biosynthetic precursor to the p-aminobenzoate (pABA) portion of folate in a process that requires the CT610 enzyme from C. trachomatis We further provide evidence that CT610 is a self-sacrificing or "suicide" enzyme that uses its own amino acid residue(s) as the substrate for pABA synthesis. This work provides the foundation for future investigation of this chlamydial pABA synthase, which could lead to new therapeutic strategies for C. trachomatis infections.

Archaeosine Modification of Archaeal tRNA: Role in Structural Stabilization.

Archaeosine (G+) is a structurally complex modified nucleoside found quasi-universally in the tRNA of Archaea and located at position 15 in the dihydrouridine loop, a site not modified in any tRNA outside the Archaea G+ is characterized by an unusual 7-deazaguanosine core structure with a formamidine group at the 7-position. The location of G+ at position 15, coupled with its novel molecular structure, led to a hypothesis that G+ stabilizes tRNA tertiary structure through several distinct mechanisms. To test whether G+ contributes to tRNA stability and define the biological role of G+, we investigated the consequences of introducing targeted mutations that disrupt the biosynthesis of G+ into the genome of the hyperthermophilic archaeon Thermococcus kodakarensis and the mesophilic archaeon Methanosarcina mazei, resulting in modification of the tRNA with the G+ precursor 7-cyano-7-deazaguansine (preQ0) (deletion of arcS) or no modification at position 15 (deletion of tgtA). Assays of tRNA stability from in vitro-prepared and enzymatically modified tRNA transcripts, as well as tRNA isolated from the T. kodakarensis mutant strains, demonstrate that G+ at position 15 imparts stability to tRNAs that varies depending on the overall modification state of the tRNA and the concentration of magnesium chloride and that when absent results in profound deficiencies in the thermophily of T. kodakarensisIMPORTANCE Archaeosine is ubiquitous in archaeal tRNA, where it is located at position 15. Based on its molecular structure, it was proposed to stabilize tRNA, and we show that loss of archaeosine in Thermococcus kodakarensis results in a strong temperature-sensitive phenotype, while there is no detectable phenotype when it is lost in Methanosarcina mazei Measurements of tRNA stability show that archaeosine stabilizes the tRNA structure but that this effect is much greater when it is present in otherwise unmodified tRNA transcripts than in the context of fully modified tRNA, suggesting that it may be especially important during the early stages of tRNA processing and maturation in thermophiles. Our results demonstrate how small changes in the stability of structural RNAs can be manifested in significant biological-fitness changes.

Biosynthesis and function of posttranscriptional modifications of transfer RNAs.

Posttranscriptional modifications of transfer RNAs (tRNAs) are critical for all core aspects of tRNA function, such as folding, stability, and decoding. Most tRNA modifications were discovered in the 1970s; however, the near-complete description of the genes required to introduce the full set of modifications in both yeast and Escherichia coli is very recent. This led to a new appreciation of the key roles of tRNA modifications and tRNA modification enzymes as checkpoints for tRNA integrity and for integrating translation with other cellular functions such as transcription, primary metabolism, and stress resistance. A global survey of tRNA modification enzymes shows that the functional constraints that drive the presence of modifications are often conserved, but the solutions used to fulfill these constraints differ among different kingdoms, organisms, and species.