Hauth, Franziskus
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Canavanine utilization and detoxification in bacteria
The nitrogen rich compound guanidine is common in nature. A variety of molecules contain a guanidino group, ranging from amino acids like arginine and canavanine to the nucleobase guanine and secondary metabolites like streptomycin. Guanidine is sensed by at least four different classes of riboswitches that are widespread in bacteria. However, limited insights into the source and physiological role of guanidine in nature exist. For this thesis, canavanine, the -oxa-analogue of arginine, was explored as a source of guanidine. Thereby, a novel canava-nine-degrading Pseudomonad was isolated and due to its ability to grow on canavanine as sole carbon and nitrogen source it was named Pseudomonas canavaninivorans. Within this work the bacterium is characterized employing polyphasic taxonomy and screened for its spe-cific phenotypic traits that allow distinguishing it from closely related type strains. Furthermore, it is presented how the bacterium circumvents the toxicity of canavanine, which originates from incorporation into proteins as well as arginase-mediated hydrolysis to canaline that forms sta-ble oximes with carbonyls. First, a specific canavanine degradation pathway is described in great detail. The key enzyme, a PLP-dependent canavanine--lyase, was subjected to exten-sive characterization and further enzyme activities that facilitate canavanine utilization are elu-cidated. In addition, the distribution of the novel pathway and its implications in nature are dis-cussed. Second, it is presented how P. canavaninivorans achieves error-free translation. The arginine-tRNA-synthetase of the bacterium is not able to discriminate between arginine and canavanine as substrates, so it uses a standalone specific canavanyl-tRNAArg editing protein to protect itself from misincorporation. We therefore propose canavanyl-tRNAArg deacylase (CtdA) as the systematic name for the editing protein. The results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in na-ture. Finally, guanidine riboswitch-associated gene functions are explained in order to get in-sights into the compound’s physiology and purpose. Not only is it shown that guanidine can be used as a nitrogen source but also guanidine riboswitches are often associated with the newly described CtdA. Beyond being used as a source of nitrogen, the findings hint towards an in-trinsic connection between canavanine and guanidine metabolism. In sum, the data suggests a paradigm shift from riboswitch-controlled detoxification of guanidine to the uptake and assimila-tion of this enigmatic nitrogen-rich compound and to its usage as an indicator molecule for guanidine-group containing molecules like canavanine.
Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent γ-lyase in Pseudomonadaceae and Rhizobiales
Canavanine, the δ-oxa-analogue of arginine, is produced as one of the main nitrogen storage compounds in legume seeds and has repellent properties. Its toxicity originates from incorporation into proteins as well as arginase-mediated hydrolysis to canaline that forms stable oximes with carbonyls. So far no pathway or enzyme has been identified acting specifically on canavanine. Here we report the characterization of a novel PLP-dependent enzyme, canavanine-γ-lyase, that catalyzes the elimination of hydroxyguanidine from canavanine to subsequently yield homoserine. Homoserine-dehydrogenase, aspartate–semialdehyde–dehydrogenase and ammonium–aspartate–lyase activities are also induced for facilitating canavanine utilization. We demonstrate that this novel pathway is found in certain Pseudomonas species and the Rhizobiales symbionts of legumes. The findings broaden the diverse reactions that the versatile class of PLP-dependent enzymes is able to catalyze. Since canavanine utilization is found prominently in root-associated bacteria, it could have important implications for the establishment and maintenance of the legume rhizosphere.
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 (Raultella 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 towards guanidine whereas the other enzyme prefers urea. We characterize the distribution of riboswitch‐associated carboxylases and Gdx exporters in bacterial habitats by analysing 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.
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.
A standalone editing protein deacylates mischarged canavanyl-tRNAArg to prevent canavanine incorporation into proteins
Error-free translation of the genetic code into proteins is vitally important for all organisms. Therefore, it is crucial that the correct amino acids are loaded onto their corresponding tRNAs. This process is highly challenging when aminoacyl-tRNA-synthetases encounter structural analogues to the native substrate like the arginine antimetabolite canavanine. To circumvent deleterious incorporation due to tRNA mischarging, editing mechanisms have evolved. However, only for half of the tRNA synthetases, editing activity is known and only few specific standalone editing proteins have been described. Understanding the diverse mechanisms resulting in error-free protein synthesis is of great importance. Here, we report the discovery of a protein that is upregulated upon canavanine stimulation in bacteria that live associated with canavanine-producing plants. We demonstrate that it acts as standalone editing protein specifically deacylating canavanylated tRNAArg. We therefore propose canavanyl-tRNAArgdeacylase (CtdA) as systematic name. Knockout strains show severe growth defects in canavanine-containing media and incorporate high amounts of canavanine into the proteome. CtdA is frequently found under control of guanidine riboswitches, revealing a functional connection of canavanine and guanidine metabolisms. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.
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.