Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803
Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803
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DOI：
10.1073/pnas.1000771107
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发表时间：
2010-09-21
影响因子：
11.1
通讯作者：
Hosaka, Hideo
中科院分区：
文献类型：
--
作者：
Kato, Kazushige;Tanaka, Ryouichi;Hosaka, Hideo
原卟啉原氧化酶（Protox）在四吡咯分子合成过程中催化原卟啉原IX氧化为原卟啉IX。原原虫在真核生物中由hemY基因编码，在许多γ -变形菌中由hemG基因编码，包括大肠杆菌。有人认为，其他细菌拥有一种尚未确定的原生细菌。为了鉴定编码Protox的独特细菌基因，我们首先将拟南芥hemY基因导入到蓝细菌Synechocystis sp. PCC6803的基因组中。随后，我们通过转座子标记对细胞进行诱变，并筛选对乙酰氟芬敏感的突变体，乙酰氟芬是hemy型Protox的特异性抑制剂。几种含有标记slr1790位点的细胞系表现出对氟虫腈的敏感性。slr1790基因编码一种假定的跨膜蛋白，该蛋白与NADH脱氢酶复合体i的M亚基有远亲关系。我们试图在聚胞菌的野生型背景下破坏该基因，但我们只能获得异质干扰物。这些细胞积累了大量的原卟啉IX，表明slr1790基因对细胞的生长和原卟啉活性至关重要。我们发现大多数蓝藻和许多其他细菌都具有slr1790同源物。我们在大肠杆菌中过表达球形红杆菌的slr1790同源物，发现该重组蛋白在体外具有原生菌活性。这些结果共同表明，slr1790编码一种独特的Protox酶，我们建议将slr1790基因命名为“hemJ”。
Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many gamma-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene "hemJ."