Guanidine production by plant homoarginine-6-hydroxylases.

Metabolism and biological functions of the nitrogen-rich compound guanidine have long been neglected. The discovery of four classes of guanidine-sensing riboswitches and two pathways for guanidine degradation in bacteria hint at widespread sources of unconjugated guanidine in nature. So far, only three enzymes from a narrow range of bacteria and fungi have been shown to produce guanidine, with the ethylene-forming enzyme (EFE) as the most prominent example. Here, we show that a related class of Fe2+- and 2-oxoglutarate-dependent dioxygenases (2-ODD-C23) highly conserved among plants and algae catalyze the hydroxylation of homoarginine at the C6-position. Spontaneous decay of 6-hydroxyhomoarginine yields guanidine and 2-aminoadipate-6-semialdehyde. The latter can be reduced to pipecolate by pyrroline-5-carboxylate reductase but more likely is oxidized to aminoadipate by aldehyde dehydrogenase ALDH7B in vivo. Arabidopsis has three 2-ODD-C23 isoforms, among which Din11 is unusual because it also accepted arginine as substrate, which was not the case for the other 2-ODD-C23 isoforms from Arabidopsis or other plants. In contrast to EFE, none of the three Arabidopsis enzymes produced ethylene. Guanidine contents were typically between 10 and 20 nmol*(g fresh weight)-1 in Arabidopsis but increased to 100 or 300 nmol*(g fresh weight)-1 after homoarginine feeding or treatment with Din11-inducing methyljasmonate, respectively. In 2-ODD-C23 triple mutants, the guanidine content was strongly reduced, whereas it increased in overexpression plants. We discuss the implications of the finding of widespread guanidine-producing enzymes in photosynthetic eukaryotes as a so far underestimated branch of the bio-geochemical nitrogen cycle and propose possible functions of natural guanidine production.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Guanidino acid hydrolysis by the human enzyme annotated as agmatinase.

Guanidino acids such as taurocyamine, guanidinobutyrate, guanidinopropionate, and guanidinoacetate have been detected in humans. However, except for guanidionacetate, which is a precursor of creatine, their metabolism and potential functions remain poorly understood. Agmatine has received considerable attention as a potential neurotransmitter and the human enzyme so far annotated as agmatinase (AGMAT) has been proposed as an important modulator of agmatine levels. However, conclusive evidence for the assigned enzymatic activity is lacking. Here we show that AGMAT hydrolyzed a range of linear guanidino acids but was virtually inactive with agmatine. Structural modelling and direct biochemical assays indicated that two naturally occurring variants differ in their substrate preferences. A negatively charged group in the substrate at the end opposing the guanidine moiety was essential for efficient catalysis, explaining why agmatine was not hydrolyzed. We suggest to rename AGMAT as guanidino acid hydrolase (GDAH). Additionally, we demonstrate that the GDAH substrates taurocyamine, guanidinobutyrate and guanidinopropionate were produced by human glycine amidinotransferase (GATM). The presented findings show for the first time an enzymatic activity for GDAH/AGMAT. Since agmatine has frequently been proposed as an endogenous neurotransmitter, the current findings clarify important aspects of the metabolism of agmatine and guanidino acid derivatives in humans.

Widespread bacterial utilization of guanidine as nitrogen source.

Guanidine is sensed by at least four different classes of riboswitches that are widespread in bacteria. However, only very few insights into physiological roles of guanidine exist. Genes predominantly regulated by guanidine riboswitches are Gdx transporters exporting the compound from the bacterial cell. In addition, urea/guanidine carboxylases and associated hydrolases and ABC transporters are often found combined in guanidine-inducible operons. We noted that the associated ABC transporters are configured to function as importers, challenging the current view that riboswitches solely control the detoxification of guanidine in bacteria. We demonstrate that the carboxylase pathway enables utilization of guanidine as sole nitrogen source. We isolated three enterobacteria (Raoultella terrigena, Klebsiella michiganensis, and Erwinia rhapontici) that utilize guanidine efficiently as N-source. Proteome analyses show that the expression of a carboxylase, associated hydrolases and transport genes is strongly induced by guanidine. Finding two urea/guanidine carboxylase enzymes in E. rhapontici, we demonstrate that the riboswitch-controlled carboxylase displays specificity toward guanidine, whereas the other enzyme prefers urea. We characterize the distribution of riboswitch-associated carboxylases and Gdx exporters in bacterial habitats by analyzing available metagenome data. The findings represent a paradigm shift from riboswitch-controlled detoxification of guanidine to the uptake and assimilation of this enigmatic nitrogen-rich compound.

Widespread bacterial lysine degradation proceeding via glutarate and L-2-hydroxyglutarate.

Lysine degradation has remained elusive in many organisms including Escherichia coli. Here we report catabolism of lysine to succinate in E. coli involving glutarate and L-2-hydroxyglutarate as intermediates. We show that CsiD acts as an α-ketoglutarate-dependent dioxygenase catalysing hydroxylation of glutarate to L-2-hydroxyglutarate. CsiD is found widespread in bacteria. We present crystal structures of CsiD in complex with glutarate, succinate, and the inhibitor N-oxalyl-glycine, demonstrating strong discrimination between the structurally related ligands. We show that L-2-hydroxyglutarate is converted to α-ketoglutarate by LhgO acting as a membrane-bound, ubiquinone-linked dehydrogenase. Lysine enters the pathway via 5-aminovalerate by the promiscuous enzymes GabT and GabD. We demonstrate that repression of the pathway by CsiR is relieved upon glutarate binding. In conclusion, lysine degradation provides an important link in central metabolism. Our results imply the gut microbiome as a potential source of glutarate and L-2-hydroxyglutarate associated with human diseases such as cancer and organic acidurias.

Synthesis of All Possible Canonical (3'-5'-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects.

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

TPP riboswitch characterization in Alishewanella tabrizica and Alishewanella aestuarii and comparison with other TPP riboswitches.

Riboswitches are located in non-coding areas of mRNAs and act as sensors of cellular small molecules, regulating gene expression in response to ligand binding. The TPP riboswitch is the most widespread riboswitch occurring in all three domains of life. However, it has been rarely characterized in environmental bacteria other than Escherichia coli and Bacillus subtilis. In this study, TPP riboswitches located in the 5' UTR of thiC operon from Alishewanella tabrizica and Alishewanella aestuarii were identified and characterized. Moreover, affinity analysis of TPP binding to the TPP aptamer domains originated from A. tabrizica, A. aestuarii, E.coli, and B. subtilis were studied and compared using In-line probing and Surface Plasmon Resonance (SPR). TPP binding to the studied RNAs from A. tabrizica and A. aestuarii caused distinctive changes of the In-line cleavage pattern, demonstrating them as functional TPP riboswitches. With dissociation constant of 2-4nM (depending on the method utilized), the affinity of TPP binding was highest in A. tabrizica, followed by the motifs sourced from A. aestuarii, E. coli, and B. subtilis. The observed variation in their TPP-binding affinity might be associated with adaptation to the different environments of the studied bacteria.

Abolishing HIV-1 infectivity using a polypurine tract-specific G-quadruplex-forming oligonucleotide.

BACKGROUND: HIV is primarily transmitted by sexual intercourse and predominantly infects people in Third World countries. Here an important medical need is self-protection for women, particularly in societies where condoms are not widely accepted. Therefore, availability of antiviral microbicides may significantly reduce sexual HIV transmission in such environments. METHODS: Here, we investigated structural characteristics and the antiviral activity of the polypurine tract (PPT)-specific ODN A, a 54-mer oligodeoxynucleotide (ODN) that has been previously shown to trigger the destruction of viral RNA genomes by prematurely activating the retroviral RNase H. The stability of ODN A and mutants thereof was tested at various storage conditions. Furthermore, antiviral effects of ODN A were analyzed in various tissue culture HIV-1 infection models. Finally, circular dichroism spectroscopy was employed to gain insight into the structure of ODN A. RESULTS: We show here that ODN A is a powerful tool to abolish HIV-1 particle infectivity, as required for a candidate compound in vaginal microbicide applications. We demonstrate that ODN A is not only capable to prematurely activate the retroviral RNase H, but also prevents HIV-1 from entering host cells. ODN A also exhibited extraordinary stability lasting several weeks. Notably, ODN A is biologically active under various storage conditions, as well as in the presence of carboxymethylcellulose CMC (K-Y Jelly), a potential carrier for application as a vaginal microbicide. ODN A's remarkable thermostability is apparently due to its specific, guanosine-rich sequence. Interestingly, these residues can form G-quadruplexes and may lead to G-based DNA hyperstructures. Importantly, the pronounced antiviral activity of ODN A is maintained in the presence of human semen or semen-derived enhancer of virus infection (SEVI; i.e. amyloid fibrils), both known to enhance HIV infectivity and reduce the efficacy of some antiviral microbicides. CONCLUSIONS: Since ODN A efficiently inactivates HIV-1 and also displays high stability and resistance against semen, it combines unique and promising features for its further development as a vaginal microbicide against HIV.

Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Repetitive nucleic acid sequences are often prone to form secondary structures distinct from B-DNA. Prominent examples of such structures are DNA triplexes. We observed that certain intrastrand triplex motifs are highly conserved and abundant in prokaryotic genomes. A systematic search of 5246 different prokaryotic plasmids and genomes for intrastrand triplex motifs was conducted and the results summarized in the ITxF database available online at http://bioinformatics.uni-konstanz.de/utils/ITxF/. Next we investigated biophysical and biochemical properties of a particular G/C-rich triplex motif (TM) that occurs in many copies in more than 260 bacterial genomes by CD and nuclear magnetic resonance spectroscopy as well as in vivo footprinting techniques. A characterization of putative properties and functions of these unusually frequent nucleic acid motifs demonstrated that the occurrence of the TM is associated with a high degree of genomic instability. TM-containing genomic loci are significantly more rearranged among closely related Escherichia coli strains compared to control sites. In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse. In conclusion we link intrastrand triplex motifs with the induction of genomic instability. We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

Interactions between Flavins and Quadruplex Nucleic Acids.

Quadruplex nucleic acids are widespread in genomes. They influence processes such as transcription, translation, replication, recombination, and the regulation of gene expression. Several synthetic ligands have been demonstrated to target quadruplex nucleic acids. However, only very few metabolites have been reported to interact with quadruplexes. In principle, an intracellular metabolite that selectively binds to four-stranded sequences could modulate quadruplex formation, stability, and thus functions in a riboswitch (or deoxyriboswitch) manner. Here we report quadruplex interactions with flavin derivatives such as FMN and FAD. The affinities were highest with parallel quadruplexes, with low (14-20 µm) dissociation constants. Taking into account combined intracellular flavin concentrations of 243 µm in E. coli, the observed interactions in principle open up the possibility of flavin levels affecting gene expression and other processes by modulating quadruplex formation.