NSRRC Activity Report 2023

Life Science 053 A minoglycoside antibiotics are effective in treating bacterial infections by binding to bacterial ribosomes, thereby hindering protein synthesis. The amine groups within aminoglycosides play crucial roles in recognizing their binding sites on ribosomes. However, pathogenic bacteria employ various drug-resistance mechanisms, such as acetylating amine groups, resulting in the inactivation of aminoglycoside antibiotics. Darwin once stated, “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.” In evolutionary competition, some bacteria employ aminoglycoside antibiotics to suppress competitors, prompting a responsive counter-strategy of amine acetylation. To prevail, bacteria evolve new modifications, such as N -formimidoylation or N -iminoacetylation, in response to this disarmament. Hence, the transformed antibiotics are no longer susceptible to inactivation. An international team which comprised Tsung-Lin Li of Academia Sinica, Yoshimistu Hamano of Fukui Prefectural University (Japan), Toru Dairi of Hokkaido University (Japan), and Chin-Yuan Chang of National Yang Ming Chiao Tung University, identified the enzyme (Orf1) that dictates the N -formimidoylation or N -iminodiacetylation transformation while elucidating the biosynthesis of the aminoglycoside antibiotic BD-12. Orf1 is the committed enzyme, as established by in vivo gene knockout and in vitro biochemical experiments ( Fig. 1 ). 1 To delve into the reaction mechanism of the enzyme at the molecular level, they determined the Orf1 apo and complex structures by leveraging TLS 15A1 , TPS 05A, and TPS 07A beamlines at the NSRRC. Aminoglycoside Modification Exploiting FAD-Dependent and Ligand-Enzyme NOS Bridge Dual Chemistry Orf1 is characterized as a noncanonical FAD-dependent N-formimidoyl/iminoacetyl synthase, employing a ligand-enzyme NOS bridge mechanism for transforming natural products, a handy valuable tool for designing next-generation antibiotics. Fig. 2 : (a) Two substrate-binding sites sit 13.5 Å apart. (b) The electron density region suggests a glycine imine adduct in a covalent linkage to C281 through an NOS bridge. (c) The arrangement between the amine group of glycylthricin and α-carbon of the iminoacetate adduct poses a trajectory appropriate for an addition reaction. [Reproduced from Ref. 1] Fig. 1 : N -formimidoylation or N -iminoacetylation was believed to follow multiple steps of reactions in one single reaction site, as does a typical FAD- dependent enzyme. [Reproduced from Ref. 1]