Selective use of aromatic amino acids by radical SAM proteins. – SelArom

Radical S-Adenosyl-L-Methionine (SAM) enzymes are capable of catalyzing chemically difficult reactions such as S atom insertions, cyclizations and unusual methylations. One-electron transfer from a conserved Fe4S4 cluster to SAM induces its cleavage and leads to the production of a 5'-deoxyadenosyl radical species. This radical species can extract a hydrogen atom from various substrates allowing for the propagation of the reactive unpaired electron. SAM can be either a cofactor or a substrate depending on whether the electron returns after the reaction to regenerate the reduced Fe4S4 cluster and the cofactor, or this is definitively consumed after the reaction.
Several radical SAM enzymes have been characterized in terms of structures, substrates and catalytic mechanism. However, no systematic study of the factors that modulate specific radical formation and subsequent product generation has been carried out. To perform such a study we have selected four radical SAM enzymes: HydG (a FeFe-hydrogenase maturase), ThiH (involved in thiamine synthesis), FO (didemethyl-hydroxy-deazariboflavin) synthase, (involved in the methanogenic F420 coenzyme synthesis) and NosL, (an enzyme involved in antibiotic synthesis), all of which bind an aromatic amino acid and catalyze the same intermediate reaction but generate widely different products. Tyrosine is the substrate of HydG, ThiH and FO-synthase whereas the substrate of NosL is tryptophan. In all cases, the radical reaction results in the cleavage of the amino acid Ca-Cß bond. One of the fragments liberated by this reaction is either dehydroglycine (ThiH and FO-synthase) or a glycyl radical (NosL and possibly HydG). HydG, ThiH and NosL subsequently use this fragment to generate their respective products: CO/CN-, the thiazole ring and the antibiotic nosiheptide. Conversely, the CofH component of FO-synthase uses the p-cresyl radical, the other product of the cleavage reaction when dehydroglycine is produced, for the synthesis of FO. Thus, p-cresol can either be a secondary, unproductive final by-product of the reaction, or an integral component of the product. In a related reaction, NosL carboxylates the tryptophan-derived fragment using the glycyl radical to generate its product.
The goal of this project is to understand how each protein channels the reaction toward a given pathway, making this radical reaction specific. Our intention is not to stop at deciphering the mechanism of one specific enzyme but, rather, to extend our analyses to extract general principles about the underlying radical-based chemistry. To achieve this goal we will develop a global approach combining biochemical, crystallographic, spectroscopic and computational techniques to identify both the active radical species involved in the reactions and the protein-substrate interactions that play key roles in orienting the selectivity.
The four partners have highly complementary competences that will be variously applied to the different target proteins: protein production and detailed functional studies with characterization of products and kinetic catalytic parameters by Partner 2; protein production, X-ray protein structure determination including complexes with substrates, inhibitors and products, time-resolved kinetic studies in the crystals and theoretical calculations on these systems by Partner 1 (a crystallization robot and automated crystallization plate monitoring setup are operational inside a glove box; this is one of the very few setups for automated anaerobic protein crystallization worldwide); Mössbauer and EPR spectroscopic experimental and theoretical studies by Partners 3 and 4 (Partner 3 has the only Mössbauer platform dedicated to biological macromolecules in France, and one of the few existing in the world; Partner 4 is a member of an “Advanced EPR” team that performs pulsed ENDOR and ESEEM experiments).