3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iβ DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3–7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are K_m^PEP 1100 ± 100 μM, K_m^E4P 530 ± 100 μM, and k_cat 10.3 ± 1.2 s⁻¹. The kinetic parameters for AroG2 are K_m^PEP 90 ± 20 μM, K_m^E4P 130 ± 40 μM, and k_cat 2.0 ± 0.2 s⁻¹. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

3-脱氧-D-阿拉伯-庚酮糖酸 7-磷酸 (DAHP) 合酶催化磷酸烯醇式丙酮酸 (PEP) 与d-赤藓糖 4-磷酸 (E4P)的缩合，并在调节细菌中芳香族氨基酸生物合成的碳通量中起重要作用和植物。来自甲醇芽孢杆菌MGA3的 DAHP 合酶 AroG1 和 AroG2 的序列分析表明这种嗜热、甲基营养细菌具有两种 Iβ 型 DAHP 合酶。本研究描述了大肠杆菌中AroG1 和 AroG2 的产生作为六组氨酸融合蛋白，通过亲和层析纯化。用 TEV 蛋白酶处理提供了用于表征和动力学分析的天然蛋白质。AroG1 和 AroG2 分别是 30.1 kDa 和 40.0 kDa 的蛋白质。两种酶在 6.3-7.2 的 pH 范围内都具有最大活性。AroG1 在 50 °C 和 pH 7.2 下的表观动力学参数为K _m ^PEP 1100 ± 100 μM、K _m ^E4P 530 ± 100 μM 和k _cat 10.3 ± 1.2 s ^-1。AroG2 的动力学参数为K _m ^PEP 90 ± 20 μM、K _m ^E4P 130 ± 40 μM 和k _cat 2.0 ± 0.2 s^-1。在50℃下后AroG2 96分钟保留其活性的50％，而AroG1 10分钟AroG2，其含有N-末端调节结构域，由分支酸和预苯酸，但不阻碍后保持其活性的小于5％的升-苯丙氨酸，l -酪氨酸，或l -色氨酸。AroG1 不受任何检查的分子的抑制。了解甲醇双歧杆菌中的DAHP 合酶调节是生成生物催化剂的第一步，该催化剂利用富含目标的芳香族氨基酸生物合成途径从甲醇合成化学品。