New enzyme identified in drug-resistant bacteria
Researchers from the Singapore-MIT Alliance for Research & Technology’s (SMART) Antimicrobial Resistance (AMR) interdisciplinary research group, together with collaborators from Massachusetts Institute of Technology (MIT), Nanyang Technological University (NTU Singapore) and institutions in the US, Poland and France, have identified amino valeramididine synthetase (AvaS), the first known pyridoxal phosphate (PLP)-dependent enzyme involved in producing a chemical modification linked to bacterial responses to metabolic stress.
The discovery provides new insight into how bacteria use RNA modifications to regulate protein production and adapt to changing conditions, including exposure to antibiotics. It could also point to new ways of studying bacterial adaptation and identifying potential targets for future antimicrobial treatments.
Antimicrobial resistance is a major global health challenge, with bacteria and other pathogens developing resistance to existing treatments and making infections harder to treat. Bacteria use several strategies to survive antibiotics, including pumping drugs out of their cells, producing enzymes that break them down and altering cellular processes targeted by the drugs.
Many of these strategies depend on the ability to regulate which proteins are produced, when they are made and how accurately genetic instructions are translated. Transfer ribonucleic acids (tRNAs) are among the RNA molecules involved in this process. They help translate genetic information into proteins and carry chemical modifications that can influence protein production under changing conditions.
In a paper titled “Pyridoxal phosphate-dependent biosynthesis of amino valeramide by AvaS in tRNA”, published in Nature Chemical Biology, the researchers identified AvaS as the enzyme responsible for producing a tRNA modification known as amino valeramide cytidine (ava²C) in Pseudomonas aeruginosa, a bacterium associated with serious infections including pneumonia and sepsis.
Ava²C had previously been detected in several bacteria and plants, but the enzyme responsible for producing it was unknown. Using SMART AMR’s high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform for RNA modification profiling, the team screened thousands of Pseudomonas aeruginosa mutants and identified AvaS. The researchers also confirmed the presence of ava²C in Acinetobacter baumannii and Vibrio cholerae, as well as in the plant Arabidopsis thaliana.
The study found that AvaS uses PLP, a derivative of vitamin B6, to convert the known modification lysidine (k²C) into ava²C. This is the first reported example of a PLP-dependent enzyme directly involved in tRNA modification. PLP-dependent enzymes have traditionally been associated with amino acid metabolism and related biochemical processes.
The discovery places PLP-dependent enzymes in a previously unrecognized class of tRNA-modifying enzymes, adding a new chemical mechanism to those already known to regulate protein production, including methylation, thiolation and isomerization. It also identifies a new biological function for PLP-dependent enzymes, showing that they can directly modify tRNA as well as participate in metabolic processes.
The researchers found that ava²C affects how bacteria read genetic codes, allowing them to produce proteins more rapidly and efficiently and adapt to metabolic and oxidative stress.
Prof Peter Dedon, Co-lead Principal Investigator at SMART AMR, Professor of Biological Engineering at MIT and co-corresponding author of the paper, said the discovery adds to growing knowledge of the functions of RNA modifications and could provide new insight into processes relevant to antimicrobial resistance and infectious disease.
Dr Jingjing Sun, Research Scientist at SMART AMR, first author and co-corresponding author of the paper, said the finding shows for the first time that PLP-dependent enzymes can directly modify tRNA, opening new areas of research into RNA chemistry and bacterial adaptation.
The SMART AMR team plans to investigate how ava²C affects bacterial stress responses and metabolism, as well as whether disrupting the modification could affect bacterial survival. Since ava²C was also found in plants, future research could examine whether similar mechanisms occur in other organisms and how the modification affects protein production.
The work also demonstrates the potential of SMART AMR’s epitranscriptomics platform for identifying previously unknown RNA-modifying enzymes on a large scale. The researchers said this could have applications in biotechnology and drug discovery, including the search for new targets for antimicrobial treatments.
The research was conducted at SMART with funding from Singapore’s National Research Foundation under its Campus for Research Excellence and Technological Enterprise (CREATE) program.
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