Backbone Thioamidation of a Ribosomal Subunit Protein in Pseudomonadota

The ribosome is an intensively studied machine responsible for protein synthesis. Recent high-resolution structures of the Escherichia coli ribosome unexpectedly revealed a thioamide on the large subunit protein uL16. This unusual and easily overlooked modification replaces oxygen with sulfur in the peptide backbone, and here, the modification is proximal to the peptidyl transferase center (PTC). The responsible enzyme has remained unidentified, although methanogenic YcaO enzymes are known to catalyze thioamidation of methyl-coenzyme M reductase. Here, we use several approaches to assign EcYcaO as the enzyme responsible for uL16 thioamidation. We began by individually predicting the structures of all E. coli proteins complexed with EcYcaO, revealing that EcuL16 was the only protein forming a high-confidence, catalytically competent interaction. Furthermore, we performed mutational analysis of the EcYcaO-EcuL16 binding interface, revealing an extensive, electrostatically complementary surface atypical of characterized YcaO enzymes. In log-phase E. coli, we observed a complex, nonlinear growth relationship between thioamidation and β-hydroxylation of EcuL16-Arg81, a neighboring PTC modification. Beyond E. coli, bioinformatics surveys predict that several thousand Pseudomonadota organisms will equivalently perform uL16 thioamidation. This prediction was validated for two Gram-negative human pathogens, Klebsiella pneumoniae and Pseudomonas aeruginosa. Overall, this work has elucidated the enzyme responsible for uL16 thioamidation and demonstrated that this unusual modification is widespread in Pseudomonadota. Further, we have laid a critical foundation for understanding both the mechanism by which this modification is formed and its functional consequences. The in silico approach leveraged here could also find broader use in identifying gene-encoded substrates for enzymes.