Researchers from Singapore-MIT Alliance for Research & Technology (SMART) Antimicrobial Resistance (AMR) group, along with collaborators from MIT, Nanyang Technological University (NTU Singapore), and institutions in the U.S., Poland, and France, have identified a new enzyme called aminovaleramididine synthetase (AvaS). This enzyme is the first known pyridoxal phosphate (PLP)-dependent enzyme responsible for modifying RNA in bacteria. This modification is linked to how bacteria adapt to stress, such as exposure to antibiotics. The discovery offers new insights into how bacteria control protein production and could lead to better strategies for combating antimicrobial resistance, a growing global health challenge. Antimicrobial resistance occurs when bacteria evolve to withstand the effects of antibiotics, making infections more difficult to treat. If left unchecked, common medical procedures and even minor injuries could become life-threatening. The identification of AvaS is significant because it is the first PLP-dependent enzyme found to modify tRNA, a type of RNA that plays a key role in protein production. This discovery provides a new way to study bacterial survival and adaptation, potentially leading to more effective treatments for drug-resistant infections. Bacteria use various strategies to resist antibiotics, including altering the production of proteins. To make these proteins, bacteria use RNA molecules, particularly transfer RNA (tRNA), which acts like a molecular courier, delivering chemical components that help in building proteins. In the study, published in Nature Chemical Biology, researchers identified AvaS as the enzyme responsible for creating a specific tRNA modification called aminovaleramide cytidine (ava²C) in Pseudomonas aeruginosa, a bacterium that causes serious infections such as pneumonia and sepsis. While this modification had been observed in other bacteria and plants, the enzyme that produces it had not been identified before. The study used a high-throughput screening method to analyze thousands of Pseudomonas aeruginosa mutants and pinpoint AvaS. The enzyme uses PLP, a form of vitamin B6, to convert another known modification into ava²C. This marks the first time a PLP-dependent enzyme has been linked to tRNA modification. Traditionally, PLP-dependent enzymes were thought to be involved only in amino acid metabolism. The discovery expands the understanding of how these enzymes function and opens new possibilities for studying bacterial adaptation and developing new antimicrobial treatments. The research also suggests that ava²C helps bacteria read genetic codes more efficiently, enabling them to produce proteins faster and adapt to stress. Scientists from SMART AMR believe this discovery could lead to more effective ways to combat drug-resistant bacteria. Future studies will explore how this modification affects bacterial metabolism and stress responses, and whether similar mechanisms exist in other organisms. This work also highlights the power of SMART AMR's epitranscriptomics platform, which can identify unknown RNA-modifying enzymes at scale, potentially supporting the development of new drugs and treatments.