NSRRC Activity Report 2023

054 NSRRC ACTIVITY REPORT 2023 The complex structures shed new light, revealing that the glycine and glycylthricin binding sites are separate and distant, ruling out the previously proposed single reaction chamber; the coupling reaction takes place at the latter site ( Fig. 2(a) ). Notably, a “nitrogen-oxygen-sulfur” covalent bond bridge between the glycine imine substrate and the sulfur atom of the enzyme cysteine was observed ( Fig. 2(b) ). This NOS bridge differs from others, occurring as a post-translational modification amid a reactive oxygen species, with a lysine residue and a cysteine residue likely involved in protein functional regulation. 2,3 In this case, the ligand-protein NOS bridge is brought about between an oxidized glycine formed at the first binding site (FAD) and an oxidized cysteine (sulfenic acid) residue at the second binding site (glycylthricin), with the oxidized glycine transported to the second binding site through a delicate intramolecular tunnel. The glycylthricin is positioned at a suitable distance and trajectory toward the glycine imine NOS bridge ( Fig. 2(c) ) for an addition reaction. The team further revealed that two amino acid residues (E312 and F316) located near the NOS bridge are crucial, as the N -formimidoylated product converts to the N -iminodiacetyl one when mutated. These two amino acids coordinate to facilitate decarboxylation and elimination reactions, resulting in bond cleavage sites at either N–O or O–S, accounting for N -formimidoylation or N -iminoacetylation. When the bond breaks between oxygen and sulfur atoms in the context of N -iminoacetylation, the sulfenic group can be regenerated by water, in contrast to that of N -formimidoylation by molecular oxygen, ready for the following reaction cycle to proceed ( Fig. 3(a) ). Finally, the team concluded that the given modifications position themselves to prevent antibiotics from inactivation by the acetylation modification of strains carrying the resistance genes ( Fig. 3(b) ), emphasizing that the modification is a convenient approach to designing the next generations of aminoglycoside antibiotics. (Reported by Yung-Lin Wang, Academia Sinica). This report features the work of Tsung-Lin Li and his collaborators published in Nat. Comum. 14 , 2528 (2023). TPS 05A Protein Microcrystallography TPS 07A Micro-focus Protein Crystallography TLS 15A1 Biopharmaceuticals Protein Crystallography • XPS, Protein Crystallography • Biological Macromolecules, Protein Structures, Life Science References 1. Y. L. Wang, C. Y. Chang, N. S. Hsu, I. W. Lo, K. H. Lin, C. L. Chen, C. F. Chang, Z. C. Wang, Y. Ogasawara, T. Dairi, C. Maruyama, Y. Hamano, T. L. Li, Nat. Comum. 14 , 2528 (2023). 2. M. Wensien, F. R. von Pappenheim, L. M. Funk, P. Kloskowski, U. Curth, U. Diederichsen, J. Uranga, J. Ye, P. Fang, K. T. Pan, H. Urlaub, R. A. Mata, V. Sautner, K. Tittmann, Nature 593 , 460 (2021). 3. F. R. von Pappenheim, M. Wensien, J. Ye, J. Uranga, I. Irisarri, J. de Vries, L. M. Funk, R. A. Mata, K. Tittmann, Nat. Chem. Biol. 18 , 368 (2022). Fig. 3 : (a) The proposed catalytic mechanism of Orf1 based on intrinsic experimental findings. (b) A strategy that antibiotic-producing species employ to counter drug resistance in competing species. [Reproduced from Ref.1]