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.
