Backbone Thioamidation of a Ribosomal Subunit Protein in Pseudomonadota

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The ribosome, crucial for protein synthesis, was found to have an unusual thioamide modification on the large subunit protein uL16 in Escherichia coli.
• This modification, replacing backbone oxygen with sulfur near the peptidyl transferase center, lacked an identified enzyme.
• Methanogenic YcaO enzymes are known to catalyze similar thioamidation reactions.

Purpose of the Study:

• To identify the enzyme responsible for the uL16 thioamidation in Escherichia coli.
• To investigate the interaction between the enzyme and its substrate.
• To determine the prevalence and potential functional significance of this modification.

Main Methods:

• In silico structure prediction of E. coli proteins interacting with EcYcaO.
• Mutational analysis of the EcYcaO-Ec uL16 binding interface.
• Bioinformatic surveys to predict the distribution of uL16 thioamidation in other organisms.

Main Results:

• EcYcaO was identified as the enzyme catalyzing uL16 thioamidation, forming a high-confidence, catalytically competent interaction.
• The binding interface revealed an extensive, electrostatically complementary surface, atypical for known YcaO enzymes.
• Thioamidation showed a complex relationship with another modification (β-hydroxylation) of uL16-Arg81.
• Bioinformatics predicted widespread uL16 thioamidation in Pseudomonadota, validated in Klebsiella pneumoniae and Pseudomonas aeruginosa.

Conclusions:

• EcYcaO is the enzyme responsible for uL16 thioamidation in E. coli.
• This unusual modification is prevalent across numerous Pseudomonadota species.
• The study provides a foundation for understanding the mechanism and functional impact of uL16 thioamidation.
• The in silico approach may aid in identifying other enzyme-substrate relationships.