Biology subjects

Kuruma, Y.

Publications and source records attributed to Kuruma, Y..

3 recordsLinked to original sources

bioRxiv2025

Phage lysis protein LysM acts as a wedge to block MurJ conformational changes

Many antibiotics target essential cellular processes. To combat multidrug-resistant bacteria, new antibacterial strategies are needed. In the peptidoglycan biogenesis pathway in Escherichia coli, MurJ, the lipid II flippase, is an essential membrane protein. The 37-residue protein M from the Levivirus phage, known as LysM or SglM, targets MurJ and induces cell lysis; however, its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the MurJ/LysM (JM) complex at 3.09 [A] resolution, revealing that LysM interacts with the crevasse between TM2 and TM7 of MurJ, locking MurJ in an outward-facing conformation, with LysM acting like a wedge. Alanine-scanning mutagenesis and pull-down assays revealed key residues responsible for LysM function, and molecular dynamics simulations showed that LysM stabilizes MurJs outward-facing state. These findings demonstrate an unprecedented phage-derived mechanism for blocking lipid II transport, providing a structural framework for designing MurJ-targeted antimicrobial agents.

molecular biology↗

bioRxiv2021

Reconstruction of phospholipid synthesis by combing in vitro fatty acid synthesis and cell-free gene expression

Phospholipid synthesis is a fundamental process that promotes cell propagation and, presently, is the most challenging issue in artificial cell research aimed at reconstituting living cells from biomolecules. Here, we constructed a cell-free phospholipid synthesis system that combines in vitro fatty acid synthesis and a cell-free gene expression system that synthesizes acyltransferases for phospholipid synthesis. Fatty acids were synthesized from acetyl-CoA and malonyl-CoA, then continuously converted into phosphatidic acids by the cell-free synthesized acyltransferases. Because the system can avoid the accumulation of synthetic intermediates that suppress the reaction, the yield of phospholipid has significantly improved from previous schemes (up to 400 {micro}M). Additionally, by adding enzymes for recycling CoA, we synthesized phosphatidic acids from acetic acid and bicarbonate as carbon sources. The constructed system is available to express the genes from pathogenic bacteria and to analyze the synthesized phospholipids. By encapsulating our system inside giant vesicles, it would be possible to construct the artificial cells in which the membrane grows and divides sustainably.

synthetic biology↗

bioRxiv2017

Uncovering cell-free protein expression dynamics by a promoter library with diverse strengths

Cell-free protein expression systems have been widely used for synthetic biology and metabolic engineering applications in recent years. Yet little is known about protein expression in the cell-free systems. Here we take a systems approach to uncover underlying dynamics of cell-free protein expression. We construct a set of T7 promoter variants to express proteins at different transcription rates in a reconstituted and E. coli extract-based cell-free systems. We find that the maximum expression level and the rate of protein synthesis as responses to the transcription rate change are different in the two cell-free systems, suggesting they are driven by different expression dynamics. We confirm this by constructing a simple mathematical model for each cell-free system, which well reproduce the experimental results and also identify different limiting factors for better protein expression in the two cell-free systems. In particular, they revealed there is a negative feedback effect in the mRNA-protein translation by the PURE system and also identified different limiting factors for better protein expression in the two cell-free systems.

synthetic biology↗