Department of Microbiology, Harvard Medical School, Boston, MA 02115.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
Proc Natl Acad Sci U S A. 2020 Nov 24;117(47):29669-29676. doi: 10.1073/pnas.2008929117. Epub 2020 Nov 10.
Carbohydrate polymers exhibit incredible chemical and structural diversity, yet are produced by polymerases without a template to guide length and composition. As the length of carbohydrate polymers is critical for their biological functions, understanding the mechanisms that determine polymer length is an important area of investigation. Most Gram-positive bacteria produce anionic glycopolymers called lipoteichoic acids (LTA) that are synthesized by lipoteichoic acid synthase (LtaS) on a diglucosyl-diacylglycerol (GlcDAG) starter unit embedded in the extracellular leaflet of the cell membrane. LtaS can use phosphatidylglycerol (PG) as an alternative starter unit, but PG-anchored LTA polymers are significantly longer, and cells that make these abnormally long polymers exhibit major defects in cell growth and division. To determine how LTA polymer length is controlled, we reconstituted LtaS in vitro. We show that polymer length is an intrinsic property of LtaS that is directly regulated by the identity and concentration of lipid starter units. Polymerization is processive, and the overall reaction rate is substantially faster for the preferred GlcDAG starter unit, yet the use of GlcDAG leads to shorter polymers. We propose a simple mechanism to explain this surprising result: free starter units terminate polymerization by displacing the lipid anchor of the growing polymer from its binding site on the enzyme. Because LtaS is conserved across most Gram-positive bacteria and is important for survival, this reconstituted system should be useful for characterizing inhibitors of this key cell envelope enzyme.
碳水化合物聚合物表现出令人难以置信的化学和结构多样性，但它们是由聚合酶在没有模板指导长度和组成的情况下产生的。由于碳水化合物聚合物的长度对其生物功能至关重要，因此了解决定聚合物长度的机制是一个重要的研究领域。大多数革兰氏阳性菌产生阴离子糖聚合物，称为脂磷壁酸（LTA），由脂磷壁酸合酶（LtaS）在细胞膜外叶嵌入的二葡萄糖基二酰基甘油（GlcDAG）起始单元上合成。LtaS 可以使用磷脂酰甘油（PG）作为替代起始单元，但 PG 锚定的 LTA 聚合物明显更长，并且制造这些异常长聚合物的细胞在细胞生长和分裂方面存在主要缺陷。为了确定 LTA 聚合物长度是如何控制的，我们在体外重新构建了 LtaS。我们表明，聚合物长度是 LtaS 的固有特性，直接受脂质起始单元的身份和浓度调节。聚合是连续的，对于首选的 GlcDAG 起始单元，整体反应速率要快得多，但 GlcDAG 的使用会导致较短的聚合物。我们提出了一个简单的机制来解释这个令人惊讶的结果：游离起始单元通过将生长聚合物的脂质锚从酶的结合位点上置换出来，从而终止聚合。由于 LtaS 在大多数革兰氏阳性菌中都保守，并且对生存至关重要，因此这个重组系统应该有助于表征这种关键细胞包膜酶的抑制剂。
