Expression of a cloned protoporphyrinogen oxidase.
Expression of a cloned protoporphyrinogen oxidase.
复制标题
DOI：
10.1016/s0021-9258(17)42182-x
复制
发表时间：
1994-01
期刊：
影响因子：
--
通讯作者：
T. Dailey;P. Meissner;H. Dailey
中科院分区：
文献类型：
--
作者：
T. Dailey;P. Meissner;H. Dailey
先前克隆的枯草芽孢杆菌的hem Y基因（Hansson，M.，和Hederstedt，L.等人（1992）J. Bacteriol. 174，8081-8093）已经在大肠杆菌中表达。表达的蛋白质已被证明是血红素生物合成途径的倒数第二个酶，原卟啉原氧化酶（EC 1.3.3.4），因此，基因命名应为hem G。这是第一次报告的表达克隆原卟啉原氧化酶从任何来源。该酶存在于可溶性细胞质组分中，因此与所有先前报道的膜结合的真核或原核原卟啉原氧化酶不同。它利用分子氧作为末端电子受体，原卟啉原IX、中卟啉原IX和粪卟啉原III作为底物。二苯醚除草剂三氟羧草醚是真核生物酶的强抑制剂，但仅具有弱抑制作用。该酶的预测分子量为51，200，这与通过高效液相色谱法和SDS-聚丙烯酰胺凝胶电泳测定的分子量很好地一致。此外，该酶在氨基末端含有一个推定的二核苷酸结合区，这与先前证明的在表征的哺乳动物酶中存在黄素部分一致。
The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.