Homologues of neisserial heme oxygenase in gram-negative bacteria:: Degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa
Homologues of neisserial heme oxygenase in gram-negative bacteria:: Degradation of heme by the product of the pigA gene of Pseudomonas aeruginosa
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DOI：
10.1128/jb.183.21.6394-6403.2001
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发表时间：
2001-11-01
影响因子：
3.2
通讯作者：
Stojiljkovic, I
中科院分区：
文献类型：
--
作者：
Ratliff, M;Zhu, WM;Stojiljkovic, I
血红素氧化裂解释放铁是一些细菌病原体利用血红素作为铁源的一种机制。铜绿假单胞菌的pigA基因编码血红素加氧酶蛋白，该基因与脑膜炎奈瑟菌的HemO具有显著的同源性（37%）。当编码奈瑟菌血红素加氧酶的基因hemO被pigA取代时，我们证明了pigA可以在功能上取代hemO，并允许奈瑟菌利用血红素。此外，铜绿假单胞菌的卡式诱变破坏pigA后，在补充了血红素的贫铁培养基中，血红素的利用出现缺陷。这种缺陷可以通过添加外源FeSO4（表明该突变体在铁代谢方面没有缺陷）和通过反式互补（来自带诱导启动子的质粒的pigA）来恢复。用离子交换色谱法纯化了PigA蛋白。用血红素重组的PigA紫外可见光谱显示了其他细菌和哺乳动物血红素加氧酶的特征。血红素- piga复合物可以在抗坏血酸存在下转化为铁胆汁素，这表明需要外源还原剂。对抗坏血酸还原产物进行酸化和高效液相色谱分析，鉴定出胆绿素ix - β的主要产物。这与先前表征的血红素加氧酶不同，其中胆绿素IX-a是典型的产物。我们得出结论，PigA是一种血红素加氧酶，可能代表一类具有新的区域特异性的这些酶。
The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation, with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-a is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.