Sawyer Anne, Bai Yu, Lu Yinghua, Hemschemeier Anja, Happe Thomas
AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44801, Bochum, Germany.
Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
Plant J. 2017 Jun;90(6):1134-1143. doi: 10.1111/tpj.13535. Epub 2017 Apr 26.
Molecular hydrogen (H ) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H production comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H -producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.
分子氢（H₂）可由绿藻中的[FeFe]氢化酶作为光合作用的直接产物产生。莱茵衣藻氢化酶HYDA1包含一个催化位点，该位点由一个与独特的2Fe亚簇相连的经典[4Fe4S]簇组成。体外研究表明，[4Fe4S]簇首先由管家FeS簇组装机制整合，随后是2Fe亚簇，其生物合成需要特定的成熟酶HYDEF和HYDG。为了研究体内的成熟过程，我们在一个氢化酶缺陷突变株中表达了来自莱茵衣藻叶绿体和核基因组的HYDA1（有和没有叶绿体转运肽），并检测了细胞内的酶促氢化酶活性以及体内H₂的产生。从叶绿体基因组表达HYDA1的转化体显示出与野生型相当的H₂产生水平，从核基因组表达全长HYDA1的转化体也是如此。相比之下，配备有靶向细胞质的HYDA1的细胞产生无活性的酶，这种酶只有在体外重新组装[4Fe4S]簇后才能被激活。这表明HYDA1 FeS簇只能由叶绿体FeS簇组装机制构建。此外，从莱茵衣藻叶绿体基因组表达细菌氢化酶基因CPI导致了H₂产生菌株，这表明结构非常不同的氢化酶可以在体内发挥HYDA1的作用，并且在莱茵衣藻中过表达外源氢化酶是可能的。所有叶绿体转化体都是稳定的，并且没有观察到HYDA1或CPI表达的毒性作用。
