Structure And Mechanism Study Of Bacteria Vitamin C Transporter ULaA

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.