Mechanism of membrane lipid control in plant pathogens unveiled by Yamagata University

Bacteria survive in fluctuating environments by altering the lipid composition of their cell membranes in response to environmental stress, yet the precise regulatory mechanisms underlying this process remain poorly understood. A research group including Graduate Student Mizuki Hoshi (at the time of the research) and Graduate Student Daiki Matsumoto of the Graduate School of Science and Engineering, Yamagata University, and Associate Professor Yasunori Watanabe of the Academic Research Institute (primarily assigned to the Faculty of Science) at Yamagata University has successfully determined the three-dimensional structure of AcvB, an enzyme involved in membrane lipid metabolism in plant-pathogenic bacteria.

The study clarified how AcvB, which is an enzyme that degrades lysyl-phosphatidylglycerol (Lys-PG), a phospholipid component of the cell membrane, recognizes its substrate and binds to the membrane to function. Furthermore, the findings indicate that AcvB works in tandem with the lipid synthase LpiA to locally regulate the concentration of Lys-PG within the membrane. The results were published in Communications Biology.

For living organisms, the cell membrane that shapes the cell plays a critical role in defending against external environmental stressors and various toxic substances. The cell membranes of certain bacteria contain Lys-PG, a positively charged phospholipid, which enhances resistance to antimicrobial peptides and other threats by altering the electrical charge of the membrane.

In plant-pathogenic bacteria, however, an excess of Lys-PG disrupts normal cellular functions and inhibits the bacteria's ability to infect host plants. Consequently, the proper regulation of Lys-PG levels by AcvB, an enzyme located in the periplasmic space that breaks down Lys-PG, is considered vital for the bacteria's infectivity. Yet, exactly how AcvB recognizes and degrades Lys-PG within the cell membrane, and how this regulatory mechanism is governed, had remained unknown.

To uncover the molecular mechanism of the membrane lipid-degrading enzyme AcvB in the plant pathogen Agrobacterium tumefaciens, the research group combined structural biology techniques with biochemical analyses.

First, they purified large quantities of AcvB as a recombinant protein from E. coli, crystallized it, and determined its three-dimensional structure using X-ray crystallography. Based on the structural data obtained, they conducted functional analyses to investigate the precise relationship between the enzyme's structure and its activity.

The structural analysis revealed that AcvB possesses a distinctive active site tailored for recognizing its substrate, Lys-PG. The environment surrounding this active site is negatively charged, creating a structure that selectively attracts the positively charged Lys-PG. This structural feature demonstrates the mechanism by which AcvB specifically recognizes Lys-PG as its substrate over other membrane lipids. Additionally, the structure of AcvB revealed a characteristic hydrophobic region protruding from the protein's surface. This structure is believed to interact directly with the cell membrane, suggesting it is a crucial element enabling AcvB to bind to and function on the membrane surface. By anchoring itself to the cell membrane surface, AcvB is thought to gain direct access to its substrate, driving the degradation reaction forward efficiently.

The researchers also focused on the possibility that AcvB does not act entirely in isolation, but functions in coordination with LpiA, the enzyme responsible for synthesizing Lys-PG. Their analysis indicated that a direct interaction between these two enzymes likely regulates Lys-PG levels within the membrane. In other words, a localized control mechanism is proposed wherein Lys-PG synthesized by LpiA is immediately degraded on-site by AcvB, locally balancing the amount of Lys-PG in that specific region of the membrane.

These results mark the first time the mechanism by which AcvB binds to the membrane surface and recognizes its substrate has been demonstrated based on its three-dimensional structure, showing a high probability that its activity is modulated through coordination with LpiA. Furthermore, the insights gained from this study indicate that bacterial cell membranes maintain a highly precise state by linking lipid synthesis and degradation through the cooperation of two enzymes. This is considered a vital mechanism allowing bacteria to adapt rapidly to environmental shifts.

The research group aims to further elucidate how bacteria tune their membrane conditions by resolving the complex structure of AcvB bound to LpiA and conducting detailed analyses of their interactions on the membrane.

Journal Information
Publication: Communications Biology
Title: Structural basis of substrate recognition and membrane association by the bacterial lysyl-phosphatidylglycerol hydrolase AcvB
DOI: 10.1038/s42003-026-10087-1