Negron Leonardo, Patchett Mark L, Parker Emily J
Institute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand.
Enzyme Res. 2011;2011:134893. doi: 10.4061/2011/134893. Epub 2011 Apr 5.
Dehydroquinate synthase (DHQS) catalyses the second step of the shikimate pathway to aromatic compounds. DHQS from the archaeal hyperthermophile Pyrococcus furiosus was insoluble when expressed in Escherichia coli but was partially solubilised when KCl was included in the cell lysis buffer. A purification procedure was developed, involving lysis by sonication at 30°C followed by a heat treatment at 70°C and anion exchange chromatography. Purified recombinant P. furiosus DHQS is a dimer with a subunit Mr of 37,397 (determined by electrospray ionisation mass spectrometry) and is active over broad pH and temperature ranges. The kinetic parameters are K(M) (3-deoxy-D-arabino-heptulosonate 7-phosphate) 3.7 μM and k(cat) 3.0 sec(-1) at 60°C and pH 6.8. EDTA inactivates the enzyme, and enzyme activity is restored by several divalent metal ions including (in order of decreasing effectiveness) Cd(2+), Co(2+), Zn(2+), and Mn(2+). High activity of a DHQS in the presence of Cd(2+) has not been reported for enzymes from other sources, and may be related to the bioavailability of Cd(2+) for P. furiosus. This study is the first biochemical characterisation of a DHQS from a thermophilic source. Furthermore, the characterisation of this hyperthermophilic enzyme was carried out at elevated temperatures using an enzyme-coupled assay.
脱氢奎尼酸合酶（DHQS）催化莽草酸途径中生成芳香族化合物的第二步反应。来自嗜热古菌激烈火球菌的DHQS在大肠杆菌中表达时不溶，但当细胞裂解缓冲液中加入氯化钾时可部分溶解。开发了一种纯化方法，包括在30°C下超声裂解，然后在70°C下热处理以及阴离子交换色谱法。纯化的重组激烈火球菌DHQS是一种二聚体，亚基分子量为37,397（通过电喷雾电离质谱法测定），在较宽的pH和温度范围内具有活性。在60°C和pH 6.8条件下，动力学参数为K(M)（3-脱氧-D-阿拉伯庚酮糖酸7-磷酸）3.7 μM，k(cat) 3.0 s⁻¹。EDTA可使该酶失活，几种二价金属离子（按有效性递减顺序）包括Cd²⁺、Co²⁺、Zn²⁺和Mn²⁺可恢复酶活性。在Cd²⁺存在下DHQS具有高活性，这在其他来源的酶中尚未见报道，可能与激烈火球菌对Cd²⁺的生物可利用性有关。本研究是首次对嗜热来源的DHQS进行生化特性分析。此外，使用酶偶联测定法在升高的温度下对这种嗜热酶进行了特性分析。
