Chua Wan-Zhen, Wong Rachel Lyn Ee, Chun Ye-Yu, Shien Nicole Ng Chyi, Su Tong, Maiwald Matthias, Chew Kean Lee, Lin Raymond Tzer-Pin, Hockenberry Alyson M, Luo Min, Sham Lok-To
Infectious Diseases Translational Research Programme and Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Pathology and Laboratory Medicine, KK Women's and Children's Hospital, Singapore, Singapore.
Sci Adv. 2025 Jan 24;11(4):eadr0162. doi: 10.1126/sciadv.adr0162.
Multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) family transporters are essential in glycan synthesis, flipping lipid-linked precursors across cell membranes. Yet, how they select their substrates remains enigmatic. Here, we investigate the substrate specificity of the MOP transporters in the capsular polysaccharide (CPS) synthesis pathway in . These capsule flippases collectively transport more than 100 types of capsule precursors. To determine whether they can substitute for one another, we developed a high-throughput approach to systematically examine nearly 6000 combinations of flippases and substrates. CPS flippases fall into three groups: relaxed, type-specific, and strictly specific. Cargo size and CPS acetylation affect transport, and we isolated additional gain-of-function flippase variants that can substitute for the peptidoglycan flippase YtgP (MurJ). We also showed that combining flippase variants in a single cassette allows various CPS precursors to be flipped, which may aid glycoengineering. This study reveals that MOP flippases exhibit broad specificity, shaping the evolution of glycan synthesis.
多药/寡糖基脂质/多糖（MOP）家族转运蛋白在聚糖合成中至关重要，可将脂质连接的前体翻转穿过细胞膜。然而，它们如何选择底物仍然是个谜。在这里，我们研究了MOP转运蛋白在荚膜多糖（CPS）合成途径中的底物特异性。这些荚膜翻转酶共同转运100多种类型的荚膜前体。为了确定它们是否可以相互替代，我们开发了一种高通量方法来系统地检测近6000种翻转酶和底物的组合。CPS翻转酶分为三组：宽松型、类型特异性和严格特异性。货物大小和CPS乙酰化会影响转运，并且我们分离出了可以替代肽聚糖翻转酶YtgP（MurJ）的其他功能获得性翻转酶变体。我们还表明，在单个盒中组合翻转酶变体可以使各种CPS前体翻转，这可能有助于糖工程。这项研究表明，MOP翻转酶具有广泛的特异性，塑造了聚糖合成的进化。
