Suh Man-Hee, Pulakat Lakshmi, Gavini Nara
Department of Biological Sciences, Bowling Green State University, Bowling Green, Ohio 43403, USA.
J Biol Chem. 2003 Feb 14;278(7):5353-60. doi: 10.1074/jbc.M208969200. Epub 2002 Dec 4.
The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.
复合金属酶固氮酶的钼铁蛋白组分是一种由nifD和nifK基因编码的α2β2四聚体。在固氮生物中，α和β亚基作为单独的多肽进行翻译，然后组装成包含两种金属中心（P簇和铁钼辅因子）的四聚体钼铁蛋白复合物。在棕色固氮菌中，铁钼辅因子生物合成位点的NifEN复合物是一种α2β2四聚体，其结构与钼铁蛋白相似，由nifE和nifN基因编码为两种单独的多肽。在多变鱼腥藻中，已表明由翻译融合的nifE和nifN基因编码的NifE-N融合蛋白能够支持生物固氮。钼铁蛋白与NifEN复合物之间的结构相似性促使我们测试钼铁蛋白以nifD-K翻译融合编码的单一蛋白形式合成时是否也具有功能。在此我们报道，棕色固氮菌中由nifD-K翻译融合编码的NifD-K融合蛋白是一种大蛋白（通过蛋白质免疫印迹分析确定），并且能够支持生物固氮。这些结果表明钼铁蛋白具有灵活性，即它能够适应主要的结构变化并保持功能。
