Institute of Low Temperature Science, Hokkaido University, N19W8, Kita-Ku, Sapporo, 060-0819, Japan.
Research Center of Inland Seas, Kobe University, Awaji, 656-2401, Japan.
J Plant Res. 2023 Jan;136(1):107-115. doi: 10.1007/s10265-022-01423-7. Epub 2022 Nov 10.
The pathways for synthesizing tetrapyrroles, including heme and chlorophyll, are well-conserved among organisms, despite the divergence of several enzymes in these pathways. Protoporphyrinogen IX oxidase (PPOX), which catalyzes the last common step of the heme and chlorophyll biosynthesis pathways, is encoded by three phylogenetically-unrelated genes, hemY, hemG and hemJ. All three types of homologues are present in the cyanobacterial phylum, showing a mosaic phylogenetic distribution. Moreover, a few cyanobacteria appear to contain two types of PPOX homologues. Among the three types of cyanobacterial PPOX homologues, only a hemJ homologue has been experimentally verified for its functionality. An objective of this study is to provide experimental evidence for the functionality of the cyanobacterial PPOX homologues by using two heterologous complementation systems. First, we introduced hemY and hemJ homologues from Gloeobacter violaceus PCC7421, hemY homologue from Trichodesmium erythraeum, and hemG homologue from Prochlorococcus marinus MIT9515 into a ΔhemG strain of E. coli. hemY homologues from G. violaceus and T. erythraeum, and the hemG homologue of P. marinus complimented the E. coli strain. Subsequently, we attempted to replace the endogenous hemJ gene of the cyanobacterium Synechocystis sp. PCC6803 with the four PPOX homologues mentioned above. Except for hemG from P. marinus, the other PPOX homologues substituted the function of hemJ in Synechocystis. These results show that all four homologues encode functional PPOX. The transformation of Synechocystis with G. violaceus hemY homologue rendered the cells sensitive to an inhibitor of the HemY-type PPOX, acifluorfen, indicating that the hemY homologue is sensitive to this inhibitor, while the wild-type G. violaceus was tolerant to it, most likely due to the presence of HemJ protein. These results provide an additional level of evidence that G. violaceus contains two types of functional PPOX.
四吡咯生物合成途径，包括血红素和叶绿素，在生物之间具有很好的保守性，尽管这些途径中的几种酶已经发生了分化。原卟啉原氧化酶（PPOX）催化血红素和叶绿素生物合成途径的最后一个共同步骤，由三个在系统发育上没有关系的基因编码，hemY、hemG 和 hemJ。所有三种同源物都存在于蓝细菌门中，显示出镶嵌的系统发育分布。此外，一些蓝细菌似乎含有两种类型的 PPOX 同源物。在三种类型的蓝细菌 PPOX 同源物中，只有 hemJ 同源物的功能已被实验验证。本研究的目的之一是通过两种异源互补系统为蓝细菌 PPOX 同源物的功能提供实验证据。首先，我们将来自 Gloeobacter violaceus PCC7421 的 hemY 和 hemJ 同源物、来自 Trichodesmium erythraeum 的 hemY 同源物以及来自 Prochlorococcus marinus MIT9515 的 hemG 同源物引入到 E. coli 的ΔhemG 菌株中。来自 G. violaceus 和 T. erythraeum 的 hemY 同源物以及来自 P. marinus 的 hemG 同源物补充了 E. coli 菌株。随后，我们试图用上述四种 PPOX 同源物取代蓝细菌 Synechocystis sp. PCC6803 的内源性 hemJ 基因。除了来自 P. marinus 的 hemG 之外，其他 PPOX 同源物取代了 Synechocystis 中 hemJ 的功能。这些结果表明，所有四种同源物都编码功能性 PPOX。用 G. violaceus hemY 同源物转化 Synechocystis 使细胞对 HemY 型 PPOX 的抑制剂 acifluorfen 敏感，表明 hemY 同源物对该抑制剂敏感，而野生型 G. violaceus 对此抑制剂耐受，这很可能是由于存在 HemJ 蛋白。这些结果提供了另一个证据，证明 G. violaceus 含有两种功能的 PPOX。
