Duewel H S, Woodard R W
Interdepartmental Program in Medicinal Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
J Biol Chem. 2000 Jul 28;275(30):22824-31. doi: 10.1074/jbc.M000133200.
The enzymes 3-deoxy-d-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and d-arabinose 5-phosphate or d-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. The recombinant KDO8PS, as isolated, displays an absorption band at 505 nm and contains approximately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subunit. EDTA inactivates the enzyme in a time- and concentration-dependent manner and eliminates the absorption at 505 nm. The addition of Cu(2+) to KDO8PS produces an intense absorption at 375 nm, while neither Co(2+) nor Ni(2+) produce such an effect. The EDTA-treated enzyme is reactivated by a wide range of divalent metal ions including Ca(2+), Cd(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+) and is reversibly inhibited by higher concentrations (>1 mm) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.
3-脱氧-D-甘露糖辛酮酸-8-磷酸合酶（KDO8PS）和3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸合酶（DAHPS）分别催化磷酸烯醇丙酮酸与5-磷酸-D-阿拉伯糖或4-磷酸-D-赤藓糖之间类似的缩合反应。虽然这两种酶促反应存在一些相似之处，但对大肠杆菌中这两种酶的经典研究表明，DAHPS是一种金属酶，而KDO8PS不需要金属。在此，我们证明，来自嗜热栖热菌的KDO8PS是一种金属酶，它是KDO8PS家族中第二个被详细表征的成员。分离得到的重组KDO8PS在505nm处有一个吸收峰，每个酶亚基分别含有约0.4当量的锌和0.2 - 0.3当量的铁。EDTA以时间和浓度依赖性方式使该酶失活，并消除505nm处的吸收峰。向KDO8PS中添加Cu(2+)会在375nm处产生强烈吸收，而Co(2+)和Ni(2+)均不会产生这种效果。经EDTA处理的酶可被多种二价金属离子重新激活，包括Ca(2+)、Cd(2+)、Co(2+)、Cu(2+)、Fe(2+)、Mg(2+)、Mn(2+)、Ni(2+)和Zn(2+)，并且会被某些金属的较高浓度（>1 mM）可逆抑制。通过等离子体质谱对该酶的几种金属形式进行分析表明，该酶每个四聚体优先结合一个、两个或四个金属离子。这些观察结果强烈表明，嗜热栖热菌KDO8PS在体内是一种金属酶，并揭示了KDO8PS和DAHPS家族之间以前未被认识到的关系。
