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Publication Abstract from PubMed

The enzyme m(1)A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNA(Leu) demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m(1)A22-tRNA methyltransferase TrmK.,Sweeney P, Galliford A, Kumar A, Raju D, Krishna NB, Sutherland E, Leo CJ, Fisher G, Lalitha R, Muthuraj L, Sigamani G, Oehler V, Synowsky S, Shirran SL, Gloster TM, Czekster CM, Kumar P, da Silva RG J Biol Chem. 2022 Jun;298(6):102040. doi: 10.1016/j.jbc.2022.102040. Epub 2022, May 17. PMID:35595101^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.