Collaborative Research: Evolution In Vitro: Structures of DAHPSynthase * KDOPSynthase Chimeras

0110877本项目侧重于结构研究，旨在揭示来自大肠杆菌的3-脱氧-D-阿拉伯-7-磷酸合成酶和3-脱氧-D-甘露-2-辛基-8-磷酸合成酶的作用和调控机制。这项工作将补充在部门同事兼联席PI Ronald Bauerle的实验室中合作进行的酶的突变和生化分析。DAHPS催化芳香族生物合成的第一步，即磷酸烯醇式丙酮酸(PEP)和D-红宝石-4-磷酸(E4P)的缩合。KDOPS催化PEP和D-阿拉伯糖-5-磷酸的缩合反应，这是脂多糖生物合成的重要步骤。这两种酶是遥远的同源物，具有相似的(β/α)8桶折叠。然而，这三种DAHPS同工酶与KDOPS的不同之处在于，它们需要二价金属才能激活，并受到反馈抑制的调节，每一种都对三种芳香氨基酸中的不同一种敏感。项目期间将致力于以下目标：(1)提纯和分析DAHPS(Phe)与Mn2+、PEP及其反馈抑制剂苯丙氨酸形成的络合物的晶体结构，并将其结构与已确定的结构[Mn*PEP]、[Mn*PEP]、[Mn*PGL]、[Pb*PEP]、[Cd*PEP]进行比较；(2)测定和提纯已结晶的DAHPS(Trp)*Mn*PEP的晶体结构；(3)如合作者M.Krauss的模拟研究所示，测定与PEP和E4P的类似物络合的DAHPS(Phe)*Mn的晶体结构；(4)结晶并测定Bauerle实验室产生的突变的DAHPS(Phe)酶的晶体结构(S)，其特征与我们的作用和调控模型不一致；以及(5)结晶并测定Bauerle实验室产生的具有酶活性的DAHPS/KDOPS嵌合体的晶体结构(S)。

0110877 This project focuses on structural studies designed to reveal the mechanisms of action and of regulation of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) and 3-deoxy-D- manno-2-octulosonate-8-phosphate synthase (KDOPS) from E. coli. This work will complement mutational and biochemical analysis of the enzymes carried out collaboratively in the laboratory of departmental colleague and co-PI, Ronald Bauerle. DAHPS catalyzes the first step of aromaticbiosynthesis, the condensation of phosphoenolpyruvate (PEP) and D-erythrose-4-phosphate (E4P). KDOPS catalyzes the condensation of PEP and D-arabinose-5-phosphate, an essential step in lipopolysaccharide biosynthesis. The two enzymes are distant homologs, possessing similar (beta/alpha)8 barrel folds. However, the three DAHPS isozymes differ from KDOPS in that they require a divalent metal for activation and are regulated by feedback inhibition, each being sensitive to a different one of the three aromatic amino acids. The following goals will be pursued during the project period: (1) to refine and analyze the crystal structure of DAHPS(Phe) complexed with Mn 2+, PEP and its feedback inhibitor, phenylalanine, and to compare the structure of this complex with those of structures already determined - [Mn*PEP], [Mn*PEP], [Mn*PGL], [Pb*PEP], and [Cd*PEP]; (2) to determine and refine the crystal structure of DAHPS(Trp)*Mn*PEP, which has already been crystallized; (3) to determine the crystal structures of DAHPS(Phe)*Mn complexed with analogs of PEP and of E4P, as indicated by the modeling studies of collaborator, M. Krauss; (4) to crystallize and determine the crystal structure(s) of mutant DAHPS(Phe) enzymes generated in Bauerle's lab whose characterictics are not consistent with our model of action and regulation; and (5) to crystallize and determine the crystal structure(s) of DAHPS/KDOPS chimeras generated in Bauerle's lab that have enzymatic activity.