Mutagenesis of hydrogenase accessory genes of Synechocystis sp PCC 6803 -: Additional homologues of hypA and hypB are not active in hydrogenase maturation
Mutagenesis of hydrogenase accessory genes of Synechocystis sp PCC 6803 -: Additional homologues of hypA and hypB are not active in hydrogenase maturation
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DOI：
10.1111/j.1742-4658.2006.05460.x
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发表时间：
2006-10-01
期刊：
影响因子：
5.4
通讯作者：
Appel, Jens
中科院分区：
文献类型：
--
作者：
Hoffmann, Doerte;Gutekunst, Kirstin;Appel, Jens
与氢化酶辅助基因同源的基因散布在蓝细菌集胞藻PCC 6803的整个基因组中。构建了hypA 1（slr 1675）、hypB 1（sll 1432）、hypC、hypD、hypE和hypF的缺失和插入突变体，这些突变体均不具有氢化酶活性。通过互补证实了各自基因参与酶的成熟。另外的同源物hypA2（sll1078）和hypB2（sll1079）的缺失对氢化酶活性没有影响。因此，hypA1和hypB1对氢化酶成熟具有特异性。我们认为hypA2和hypB2参与了不同的金属插入过程。添加镍可提高Delta hypA1和Delta hypB1的氢化酶活性，表明HypA1和HypB1参与镍插入到酶的活性位点中。所有的hypA和hypB单突变体和双突变体的脲酶活性与野生型细胞相同。因此，在集胞藻的氢酶和脲酶成熟过程中，这两个hyp基因似乎没有共同的功能。在全基因组中的相似性搜索产生了Slr1876作为氢化酶特异性蛋白酶的最佳候选物。相应的缺失突变体没有氢化酶活性。缺失hupE对氢化酶活性没有影响，但导致突变体不能在含有金属螯合剂次氮基三乙酸盐的培养基中生长。在添加钴或蛋氨酸后恢复生长。因为后者是由集胞藻中的钴需要酶合成的，所以HupE是蓝细菌中钴转运蛋白的良好候选者。
Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of Delta hypA1 and Delta hypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.