Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States.
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48105-1065, United States.
Bioorg Chem. 2012 Feb;40(1):79-86. doi: 10.1016/j.bioorg.2011.09.002. Epub 2011 Sep 17.
The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.
莽草酸生物合成途径中的第一个酶，3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶（DAH7PS），在表达它的细菌和植物中的大小和复杂性差异很大。古生菌 Aeropyrum pernix 的 DAH7PS（DAH7PS（Ap））是 DAH7PS 酶中最小和最简单的之一，这导致了 DAH7PS（Ap）不会受到莽草酸途径产物反馈调节的假设。我们在大肠杆菌中过表达了 DAH7PS（Ap），纯化了它，并对其酶活性进行了表征。然后，我们用二价锰离子和磷酸烯醇丙酮酸结合物（PDB ID：1VS1）解决了它的 X 射线晶体结构。DAH7PS（Ap）在溶液中是一种同二聚体金属酶。其酶活性在 60°C 以上急剧增加，最佳活性在 95°C。其在 60°C 时的 pH 最佳值为 5.7。DAH7PS（Ap）在 60°C 时遵循米氏动力学，对赤藓糖 4-磷酸的 K（M）为 280 μM，对磷酸烯醇丙酮酸的 K（M）为 891 μM，k（cat）为 1.0 s（-1）。我们测试的莽草酸生物合成途径的下游产物都没有抑制 DAH7PS（Ap）的活性。DAH7PS（Ap）的结构与 Thermatoga maritima 的 DAH7PS（PDB ID：3PG8）和 Pyrococcus furiosus 的 DAH7PS（PDB ID：1ZCO）的结构相似，与其被指定为未调节的 DAH7PS 一致。
