Discovery of novel orphan riboswitch ligands

Project Information

RiboDisc

Grant agreement ID: 681777

DOI

10.3030/681777

Project closed

EC signature date 27 March 2016

Start date 1 April 2016

End date 30 September 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 918 600,00

EU contribution

€ 1 918 600,00

Coordinated by

UNIVERSITAT KONSTANZ
Germany

Periodic Reporting for period 4 - RiboDisc (Discovery of novel orphan riboswitch ligands)

Reporting period: 2020-10-01 to 2022-09-30

Summary of the context and overall objectives of the project

Riboswitches are mRNA-based gene-regulatory elements triggered by direct interactions with small molecular ligands. They are exciting targets for novel antibiotic strategies. Many putative riboswitches have been identified using bioinformatics. However, ligand identification is getting more complicated since many motifs are not expected to be involved in simple feedback regulation mechanisms. For such “classical” riboswitches ligands have been assigned in the past based on testing metabolites selected by educated guesses guided by the associated gene contexts. We are convinced that this approach limits the identification of riboswitches that play regulatory roles in more complex bacterial processes such as virulence, detoxification, communication, and life style adaptations. Within this project, new riboswitch classes will be identified and characterized, paving the way for the development of antibiotics with novel modes of action. We will establish a systematic, robust and unbiased approach for identifying intracellular RNA ligands by fishing small molecules from lysates as well as screening fractionated cellular extracts. The methodology shows great potential for assigning novel riboswitch classes. The proposed research is highly relevant for one of the major biomedical challenges of the coming decades: Since riboswitches are effective antibacterial targets, the identification of novel riboswitch / ligand interactions has immediate implications for establishing future antibiotic strategies necessary to keep in check the progressing problem of bacterial drug resistance.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The main goal of identifying novel riboswitch ligands has been achieved with success, as exemplified in Lenkeit et al., NAR 2020. Afterwards, we focused on the investigation of gene functions that are found under control of guanidine riboswitches. Since guanidine is still a blank spot on the metabolic map, studying riboswitch-associated activities should provide vauable insights into the biology and biochemistry of guanidine in nature. In the following, the individual successes are listed, for scientific details please consult the mentioned publications:

- Novel cyclic dinucleotides were synthetized and tested as riboswitch ligands (C. Wang, M. Sinn, J. Stifel, A. C. Heiler, A. Sommershof, and J. S. Hartig: Synthesis of All Possible Canonical (3´-5´-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects. Journal of the American Chemical Society, 2017, 139, 16154)

- A novel lysine degradation pathway was discovered by serendipity and characterized while investigating metabolic aspects of guanidine riboswitches (S. Knorr, M. Sinn, D. Galetskiy, R. M. Williams, C. Wang, N. Müller, O. Mayans, D. Schleheck, and J. S. Hartig: Widespread bacterial lysine degradation proceeding via glutarate and L-2-hydroxyglutarate. Nature Communications, 2018, 9, 5071
).

- A novel guanidine riboswitch was discovered and characterized (F. Lenkeit, I. Eckert, J. S. Hartig, and Z. Weinberg: Discovery and Characterization of a Fourth Class of Guanidine Riboswitches. Nucleic Acids Research, 2020, 16, 12889) This discovery enabled us to study further riboswitch-associated genes regarding its function, see the following publications.

- Guanidine degradation for assimilation via carboxylation was studied in detail (M. Sinn, F. Hauth, F. Lenkeit, Z. Weinberg, and J. S. Hartig: Widespread bacterial utilization of guanidine as nitrogen source. Molecular Microbiology, 2021, doi: 10.1111/mmi.14702).

- A second guanidine degradation pathway via direct hydrolysis was discovered. Surprisingly, the enzyme utilizes Ni ions for catalysis (D. Funck, M. Sinn, J. Fleming, M. Stanoppi, J. Dietrich, R. López-Igual, O. Mayans, and J. S. Hartig: Discovery of a Ni2+-dependent guanidine hydrolase in bacteria. Nature, 2022, 603, 515).

- We have studied the degradation of canavanine that yields hydroxyguanidine that also serves as riboswitch ligand. For the purpose, we described and named a new bacterium (F. Hauth, H. Buck, and J. S. Hartig: Pseudomonas canavaninivorans sp. nov. isolated from bean rhizosphere. International Journal of Systematic and Evolutionary Microbiology, 2022, doi: 10.1099/ijsem.0.005203). In addition, we clarified the molecular details of the enzymatic degradation pathway in P. canavaninivorans (F. Hauth, H. Buck, M. Stanoppi and J. S. Hartig
Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent g-lyase in Pseudomonadaceae and Rhizobiales. RSC Chemical Biology, 2022, DOI: 10.1039/D2CB00128D)

- We characterized a human enzyme that was believed to be an agmatinase. Instead, we found that it hydrolyzes certain guanidinoacids while rejecting agmatine and arginine as substrates (M. Sinn, M. Stanoppi, F. Hauth, J. R. Fleming, D. Funck, O. Mayans, J. S. Hartig: Guanidino acid hydrolysis by the human enzyme annotated as agmatinase, under revision at Scientific Reports).

- We studied a further riboswitch-associated gene function, namely the B3/4-domain protein. We discovered that the gene encodes an editing enzyme that corrects arginine tRNAs mischarged with canavanine (F. Hauth, D. Funck, J.S. Hartig: A standalone editing protein deacylates mischarged canavanyl tRNAArg to prevent canavanine incorporation into proteins. under revision, Nucleic Acids Research).

- We identified a variation of the guanidine class IV riboswitch previously discovered by us (F. Lenkeit, I. Eckert, M. Sinn, F. Hauth, J. S. Hartig, and Z. Weinberg: A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity, under revision at RNA Biology).

Taken together, the project has shed significant light on the biology and biochemistry of guanidine and the associated riboswitches.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

See above

Guanidine in the active site of guanidine hydrolase, see Funck et al., Nature 2022