Notheis C, Drewke C, Leistner E
Institut für Pharmazeutische Biologie, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany.
Biochim Biophys Acta. 1995 Mar 15;1247(2):265-71. doi: 10.1016/0167-4838(94)00235-9.
The E. coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al. (Kohara, Y, Akiyama, K. and Isono K. (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E. coli chromosome. The gene was overexpressed as a MBP/PdxH fusion protein. The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography. Both the MBP/PdxH fusion protein and the PdxH protein were characterized. Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate. Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates. The enzymes did not accept non-phosphorylated substrates. Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor. As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer. No indication was found that the PdxH protein forms a dimer. The monomer was shown to be catalytically active.
编码吡哆醇 - 5'-磷酸：氧氧化还原酶（脱氨基）（EC 1.4.3.5，PdxH）的大肠杆菌基因pdxH被克隆，定位到小原等人文库（小原洋、秋山健、矶野克。（1987年）《细胞》50卷，495 - 508页）中的噬菌体20B5，并被定位到大肠杆菌染色体36.0至36.1分钟之间的一段区域。该基因作为MBP/PdxH融合蛋白过表达。融合蛋白通过在直链淀粉树脂上的亲和层析进行纯化，并在蛋白酶“因子Xa”存在下进行水解，经过另一轮柱层析后得到均一的PdxH蛋白。对MBP/PdxH融合蛋白和PdxH蛋白都进行了表征。两种酶都是依赖黄素单核苷酸的酶，在氧气存在下将磷酸吡哆醇和磷酸吡哆胺氧化为磷酸吡哆醛。两种蛋白对两种底物的米氏常数相似，范围在350至400微摩尔。这些酶不接受非磷酸化底物。动力学数据表明，该酶（MBP/PdxH）受到磷酸吡哆醛作为混合型抑制剂的产物抑制（抑制常数8微摩尔）。凝胶排阻色谱显示，融合蛋白的一小部分形成了二聚体，而大部分蛋白是单体。未发现PdxH蛋白形成二聚体的迹象。已证明单体具有催化活性。
