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.