许多细菌包括大肠杆菌在内的肠道菌等在厌氧条件下通过磷酸烯醇丙酮酸盐依赖磷酸转移酶系统（PTS）利用维生素C（又称抗坏血酸）作为碳源。PTS系统是原核生物特有的主要用于摄取碳水化合物的系统，通过催化碳水化合物磷酸化来驱动对碳水化合物的跨膜运输。 特异性转运维生素C的PTS系统属于PTS-AG超家族包括酶EI、 热稳定的磷酸载体蛋白HPr和酶EII三个部分。其中酶EI、HPr蛋白是细菌中PTS系统共用的，而酶EII部分是一个复合物，由膜上的EIIC组分、胞内的EIIB组分和EIIA组分组成，具有底物特异性。转运维生素C的酶EII包括UlaA蛋白（EIIC）、UlaB蛋白（EIIB）和 UlaC（EIIA）蛋白。 PTS系统是一个多组分转运体系，对维生素C的跨膜转运主要是由UlaA蛋白介导的。UlaA蛋白将维生素C从胞外结合转运至胞内侧时由UlaB磷酸化而释放到胞质中，其中磷酸基团是由烯醇式丙酮酸（PEP）提供，经由酶EI、HPr蛋白、UlaC蛋白和UlaB蛋白依次传递最后传递给维生素C。 为了进一步理解PTS-AG超家族的转运机制，我们解析了UlaA蛋白多个构象的结构，分别为大肠杆菌UlaA蛋白结合了维生素C的两个构象晶体结构，其中一个为朝外开口构象，分辨率1.65?；另一个为封闭构象，分辨率为2.35?，和巴斯德杆菌UlaA蛋白朝内开口构象的晶体结构，分辨率为3.3?。我们解析的结构显示UlaA蛋白是一个同源二聚体， 展现出一种全新的折叠方式，每个单体蛋白包括11个跨膜片段，分为‘V-motif’结构域和‘Core’结构域。通过对三种构象结构分析，发现维生素C完全结合在‘Core’结构域中，通过‘Core’结构域相对于‘V-motif’结构域的一个纵向刚体运动来将维生素C从胞外转运至胞内侧，从而我们确定了UlaA蛋白对维生素C的转运是一种电梯轿厢式交替开放机制。 我们解析了UlaA蛋白三个关键构象结构基本完成了PTS-AG家族成员对维生素C转运的周期过程并确定了是一种电梯轿厢式交替开放机制，加深了对PTS系统转运机制的理解。
Various bacteria can ferment vitamin C (L-ascorbate) under anaerobic conditions via the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The PTS is a multiple component carbohydrate uptake system that drives specific sugar across the bacterial inner membrane while catalyzing sugar phosphorylation. The ascorbate-specific PTS, belongs to the PTS-AG superfamily, includes an enzyme I, a heat-stable phosphocarrier protein (HPr) and an emzyme II (EII) complex. A transporter (UlaA), an emzyme IIB-like enzyme (UlaB) and an emzyme IIA-like enzyme (UlaC) forming the emzyme II (EII) complex that are sugar specific. The mechanism of PTS-mediated sugar uptake couples sugar transport to sugar phosphorylation in a “group translation” process. The UlaABC proteins as well as the energy-coupling PTS proteins including enzyme I and HPr are required for the anaerobic uptake of vitamin C and its phosphorylation to L-ascorbate 6-phosphate.The phosphate group originating from phosphoenolpyruvate (PEP) is transferred from EI to HPr, EIIA, and EIIB sequentially. Finally, EIIB transfers its phosphate group to the transported sugar bound to the membrane-spanning EIIC, and the phosphorylated sugar is then released into the cytoplasm. To further understand the mechanism of PTS-AG superfamily，we determined the crystal structures of vitamin C-bound UlaA from Escherichia coli in an outward-open and occluded conformation at 1.65 ? and 2.35 ? resolution and and the crystal structure of vitamin C-bound EIICasc component from Pasteurella multocida in the inward-facing conformation at 3.3 ? resolution. UlaA forms a homodimer and exhibits a new fold. Each UlaA protomer consists of 11 transmembrane segments arranged into a ‘V-motif’ domain and a ‘Core’ domain. By comparing three conformational states, the ascorbate translocation can be achieved by a rigid-body movement of the substrate-binding Core domain relative to the V motif domain, confirms an elevator car mechanism. The three conformational structures completes the transport cycle of PTS-AG family, provides mechanistic insights into PTS system.
