DltB, a model member of the Membrane-Bound O-AcylTransferase (MBOAT) superfamily, plays a crucial role in D-alanylation of the lipoteichoic acid (LTA), a significant component of the cell wall of gram-positive bacteria. This process stabilizes the cell wall structure, influences bacterial virulence, and modulates the host immune response. Despite its significance, the role of DltB is not well understood. Through biochemical analysis and cryo-EM imaging, we discover that Streptococcus thermophilus DltB forms a homo-tetramer on the cell membrane. We further visualize DltB in an apo form, in complex with DltC, and in complex with its inhibitor amsacrine (m-AMSA). Each tetramer features a central hole. The C-tunnel of each protomer faces the intratetramer interface and provides access to the periphery membrane. Each protomer binds a DltC without changing the tetrameric organization. A phosphatidylglycerol (PG) molecule in the substrate-binding site may serve as an LTA carrier. The inhibitor m-AMSA bound to the L-tunnel of each protomer blocks the active site. The tetrameric organization of DltB provides a scaffold for catalyzing D-alanyl transfer and regulating the channel opening and closing. Our findings unveil DltB’s dual function in the D-alanylation pathway, and provide insight for targeting DltB as a anti-virulence antibiotic.

DltB 是膜结合 O-酰基转移酶 (MBOAT) 超家族的模型成员，在脂磷壁酸 (LTA)（革兰氏阳性菌细胞壁的重要组成部分）的 D-丙氨酰化中发挥着至关重要的作用。这个过程稳定细胞壁结构，影响细菌毒力，并调节宿主免疫反应。尽管 DltB 很重要，但其作用尚不清楚。通过生化分析和冷冻电镜成像，我们发现嗜热链球菌DltB 在细胞膜上形成同源四聚体。我们进一步观察了载脂蛋白形式的 DltB、与 DltC 的复合物以及与其抑制剂安吖啶 (m-AMSA) 的复合物。每个四聚体都有一个中心孔。每个原聚体的 C 通道面向四聚体内界面并提供通向外围膜的通道。每个原聚体结合一个 DltC，而不改变四聚体组织。底物结合位点中的磷脂酰甘油 (PG) 分子可以充当 LTA 载体。与每个原聚体的 L 通道结合的抑制剂 m-AMSA 会阻断活性位点。 DltB 的四聚体组织提供了催化 D-丙氨酰转移和调节通道打开和关闭的支架。我们的研究结果揭示了 DltB 在 D-丙氨酰化途径中的双重功能，并为将 DltB 作为抗毒力抗生素提供了见解。