Researchers from the Singapore-MIT Alliance for Research & Technology (SMART) Antimicrobial Resistance (AMR) interdisciplinary research group, working with collaborators from MIT, Nanyang Technological University, and institutions in the United States, Poland, and France, have discovered aminovaleramididine synthetase (AvaS) — the first identified pyridoxal phosphate (PLP)-dependent enzyme responsible for producing a chemical modification linked to how bacteria respond to metabolic stress. The discovery sheds new light on how bacteria use RNA modification to control protein production, opening new avenues for studying bacterial adaptation and identifying future targets for antimicrobial therapeutics.

Antimicrobial resistance is one of the most pressing global health challenges of our time. Bacteria and other pathogens are rapidly developing resistance to existing treatments, making infections harder to treat — without new approaches, routine surgeries or even a paper cut could become life-threatening. Bacteria develop that resistance through various strategies, many of which depend on their ability to regulate which proteins are made, when, and how accurately, whether by pumping drugs out of their cells, breaking drugs down with enzymes, or developing new processes to dodge a drug’s target altogether.

Chemical Stickers on Delivery Vehicles

To build proteins, bacteria rely on transfer RNA (tRNA) — specialized molecules that act as delivery vehicles, bringing chemical “stickers” that help bacteria control how proteins are made in response to stress and changing conditions, including exposure to antibiotics. In a paper published in Nature Chemical Biology, the researchers describe how they identified AvaS as the enzyme responsible for creating a tRNA modification known as aminovaleramide cytidine (ava₂C) in Pseudomonas aeruginosa, a harmful bacterium behind a range of serious human infections such as pneumonia and sepsis. The modification had previously been detected in several bacteria and plants, but the enzyme responsible for producing it was unknown.

Using SMART AMR’s high-throughput mass spectrometry-based RNA modification profiling platform, the team systematically screened thousands of Pseudomonas aeruginosa mutants and discovered AvaS. They also confirmed the presence of ava₂C in other organisms, including the bacteria Acinetobacter baumannii and Vibrio cholerae, as well as the plant Arabidopsis thaliana.

The research revealed that AvaS uses PLP, a vitamin B6 derivative, to convert a known modification, lysidine (k₂C), into ava₂C — marking the first time a PLP-dependent enzyme has been linked to tRNA modification. Traditionally, PLP-dependent enzymes have only been associated with amino acid metabolism and related biochemical pathways, so the finding expands the known chemical mechanisms, alongside methylation, thiolation, and isomerization, that bacteria use to regulate protein production. The team also found that ava₂C changes how bacteria read genetic codes, allowing them to produce protein faster and more efficiently while helping them adapt to metabolic and oxidative stress.

A New Chapter in RNA Biology

“While many RNA modifications have been known for decades, researchers are still uncovering the full extent of their roles. The discovery of AvaS opens a previously unknown chapter in RNA biology and is an important step forward in our understanding of processes relevant to antimicrobial resistance,” said Prof Peter Dedon, Co-lead Principal Investigator at SMART AMR, Professor of Biological Engineering at MIT, and co-corresponding author of the paper. “As we continue to map the RNA modification landscape, we expect many more discoveries with meaningful implications for infectious disease, antimicrobial resistance, and fundamental biology.”

“Our discovery has revealed, for the first time, that PLP-dependent enzymes can directly modify tRNA, expanding our knowledge and understanding of RNA-modifying chemistry,” added Dr Jingjing Sun, Research Scientist at SMART AMR, first author and co-corresponding author of the paper. “This opens up new avenues for studying bacterial adaptation and developing new and more effective strategies to overcome drug-resistant bacteria.”

Building on this discovery, the SMART AMR team plans to investigate how ava₂C affects bacterial stress responses and metabolism, and to explore how the modification might be disrupted or prevented. With ava₂C also observed in plants, future studies could explore whether other living organisms use similar biological tools, and how the modification influences protein building beyond bacteria. More broadly, the work highlights the strength of SMART AMR’s epitranscriptomics platform as an engine for discovering unknown RNA-modifying enzymes at scale, a capability that could help pharmaceutical researchers find new drug targets as bacteria continue to develop resistance against existing treatments.

The research, published in Nature Chemical Biology, was conducted at SMART and is supported by the National Research Foundation (NRF) Singapore under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.