RiboDisc - Discovery of novel orphan riboswitch ligands
- FB Chemie
|(2022): Discovery of a Ni2+-dependent guanidine hydrolase in bacteria Nature. Springer Nature. 2022, 603(7901), pp. 515-521. ISSN 0028-0836. eISSN 1476-4687. Available under: doi: 10.1038/s41586-022-04490-x
Nitrogen availability is a growth-limiting factor in many habitats1, and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogen-rich compound guanidine occurs widely in nature2-4, but its utilization is impeded by pronounced resonance stabilization5, and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni2+-dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni2+ instead of the typical Mn2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.
|(2022): Guanidino acid hydrolysis by the human enzyme annotated as agmatinase Scientific Reports. Springer Nature. 2022, 12, 22088. eISSN 2045-2322. Available under: doi: 10.1038/s41598-022-26655-4
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.
|(2020): Discovery and characterization of a fourth class of guanidine riboswitches Nucleic Acids Research. Oxford University Press. 2020, 48(22), pp. 12889-12899. ISSN 0305-1048. eISSN 1362-4962. Available under: doi: 10.1093/nar/gkaa1102
Riboswitches are RNAs that specifically sense a small molecule and regulate genes accordingly. The recent discovery of guanidine-binding riboswitches revealed the biological significance of this compound, and uncovered genes related to its biology. For example, certain sugE genes encode guanidine exporters and are activated by the riboswitches to reduce toxic levels of guanidine in the cell. In order to study guanidine biology and riboswitches, we applied a bioinformatics strategy for discovering additional guanidine riboswitches by searching for new candidate motifs associated with sugE genes. Based on in vitro and in vivo experiments, we determined that one of our six best candidates is a new structural class of guanidine riboswitches. The expression of a genetic reporter was induced 80-fold in response to addition of 5 mM guanidine in Staphylococcus aureus. This new class, called the guanidine-IV riboswitch, reveals additional guanidine-associated protein domains that are extremely rarely or never associated with previously established guanidine riboswitches. Among these protein domains are two transporter families that are structurally distinct from SugE, and could represent novel types of guanidine exporters. These results establish a new metabolite-binding RNA, further validate a bioinformatics method for finding riboswitches and suggest substrate specificities for as-yet uncharacterized transporter proteins.
|(2020): Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria
Novel insights into amino-acid catabolism and the characterisation of a ncRNA regulating BCAA biosynthesis in bacteria
Amino acid metabolism is not only of interest because of its impact on protein biosynthesis as the motor for catalytic processes in the cell, but also catabolism of amino acids themselves presents a very important biochemical process in bacterial metabolism. Bacteria can exploit amino acids as alternative carbon sources by directly utilizing the carbon backbone for energy metabolism. Furthermore, amino acid degradation presents an important defense strategy against various stress factors. However, white spots on the map still exist for many bacterial degradation pathways of amino acids. For example, complete catabolic pathways of the basic amino acid lysine, the branched chain amino acids and most of the aromatic amino acids are still missing for E. coli, the best studied organism. This thesis presents the complete characterisation of a novel lysine degradation pathway in E. coli which can also be found in other bacterial species. We discovered a putative αketoglutarate dependent dioxygenase, CsiD, that hydroxylates glutarate producing succinate thus connecting lysine degradation to central carbon metabolism. Furthermore, we showed that the reaction product of CsiD, L-2-hydroxyglutarate, is further metabolised via LhgO to channel electrons into the membrane for respiratory energy metabolism. We present evidence that lysine is degraded, in E. coli, under nutrient starvation conditions contributing to bacterial adaptation to stationary phase conditions. Furthermore, because of the association of L-2-hyroxyglutarate to human diseases such as cancer and organic acidurias, we discuss a potential relation between the human gut microbiome as a potential source of the oncometabolite. To shed light on other possibly unknown amino acid degradation pathways in E. coli, this thesis contains the implementation of a screening assay for the identification of novel enzyme functions involved in amino acid catabolism. The screening results offer new insights into what processes influence the utilization of amino acids and derived metabolites. Different RNA based regulatory elements, like transcription attenuators and riboswitches, can be found in bacteria regulating important processes in amino acid metabolism.
|(2018): Widespread bacterial lysine degradation proceeding via glutarate and L-2-hydroxyglutarate Nature Communications. 2018, 9(1), 5071. eISSN 2041-1723. Available under: doi: 10.1038/s41467-018-07563-6
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.
|01.04.2016 – 31.03.2021