Hansson M, Gustafsson M C, Kannangara C G, Hederstedt L
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark.
Biochim Biophys Acta. 1997 Jun 20;1340(1):97-104. doi: 10.1016/s0167-4838(97)00030-7.
Oxidation of coproporphyrinogen III to coproporphyrin III is found in extracts of Escherichia coli cells containing the Bacillus subtilis HemY protein (M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962-5970). We have analysed whether this activity is due to the heterologous expression system, since it in vivo would lead to disruption of the heme biosynthetic pathway. B. subtilis hemY was fused in its 3'-end to a polynucleotide encoding six histidine residues and expressed from plasmids in both E. coli and B. subtilis. The His6-tagged HemY protein extracted from membranes using non-ionic detergent was purified by Ni2+ affinity chromatography. Isolated HemY fusion protein synthesised in E. coli and B. subtilis oxidised coproporphyrinogen III to coproporphyrin III. No direct formation of protoporphyrin IX from coproporphyrinogen III could be detected. Our results suggest that the coproporphyrinogen III to coproporphyrin III activity of HemY is either avoided in B. subtilis in vivo or that coproporphyrin III is a heme biosynthetic intermediate in this bacterium.
在含有枯草芽孢杆菌HemY蛋白的大肠杆菌细胞提取物中发现了粪卟啉原III氧化为粪卟啉III的现象（M. 汉森和L. 赫德施泰特，《细菌学杂志》176, 5962 - 5970）。我们分析了这种活性是否归因于异源表达系统，因为在体内它会导致血红素生物合成途径的中断。枯草芽孢杆菌hemY在其3'端与编码六个组氨酸残基的多核苷酸融合，并在大肠杆菌和枯草芽孢杆菌中从质粒表达。使用非离子洗涤剂从膜中提取的His6标签HemY蛋白通过Ni2 +亲和层析纯化。在大肠杆菌和枯草芽孢杆菌中合成的分离的HemY融合蛋白将粪卟啉原III氧化为粪卟啉III。未检测到粪卟啉原III直接形成原卟啉IX。我们的结果表明，HemY的粪卟啉原III到粪卟啉III的活性在枯草芽孢杆菌体内要么被避免，要么粪卟啉III是该细菌血红素生物合成的中间体。
