Hoffmann Dörte, Gutekunst Kirstin, Klissenbauer Monika, Schulz-Friedrich Rüdiger, Appel Jens
Botanisches Institut, Christian-Albrechts University, Kiel, Germany.
FEBS J. 2006 Oct;273(19):4516-27. doi: 10.1111/j.1742-4658.2006.05460.x.
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 DeltahypA1 and DeltahypB1 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.
与氢化酶辅助基因同源的基因分散在蓝藻集胞藻PCC 6803的整个基因组中。构建了hypA1（slr1675）、hypB1（sll1432）、hypC、hypD、hypE和hypF的缺失和插入突变体，这些突变体均无氢化酶活性。通过互补实验证实了各个基因参与了该酶的成熟过程。额外的同源基因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ΔhypA1和ΔhypB1的氢化酶活性，这表明HypA1和HypB1参与将镍插入酶的活性位点。所有hypA和hypB单突变体及双突变体的脲酶活性与野生型细胞相同。因此，在集胞藻中，这两个hyp基因在氢化酶和脲酶成熟过程中似乎没有共同功能。在整个基因组中进行相似性搜索，结果显示Slr1876是氢化酶特异性蛋白酶的最佳候选基因。相应的缺失突变体没有氢化酶活性。hupE的缺失对氢化酶活性没有影响，但导致突变体无法在含有金属螯合剂次氮基三乙酸的培养基中生长。添加钴或蛋氨酸后恢复生长。因为后者是集胞藻中一种需要钴的酶合成的，所以HupE是蓝藻中钴转运蛋白的良好候选基因。
