Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM), Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Pozuelo de Alarcón, Madrid, 28223, Spain.
Departamento de Biotecnología-Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaría y de Biosistemas, UPM, Madrid, Spain.
Microb Biotechnol. 2021 May;14(3):1073-1083. doi: 10.1111/1751-7915.13758. Epub 2021 Jan 28.
Engineering nitrogenase in eukaryotes is hampered by its genetic complexity and by the oxygen sensitivity of its protein components. Of the three types of nitrogenases, the Fe-only nitrogenase is considered the simplest one because its function depends on fewer gene products than the homologous and more complex Mo and V nitrogenases. Here, we show the expression of stable Fe-only nitrogenase component proteins in the low-oxygen mitochondria matrix of S. cerevisiae. As-isolated Fe protein (AnfH) was active in electron donation to NifDK to reduce acetylene into ethylene. Ancillary proteins NifU, NifS and NifM were not required for Fe protein function. The FeFe protein existed as apo-AnfDK complex with the AnfG subunit either loosely bound or completely unable to interact with it. Apo-AnfDK could be activated for acetylene reduction by the simple addition of FeMo-co in vitro, indicating preexistence of the P-clusters even in the absence of coexpressed NifU and NifS. This work reinforces the use of Fe-only nitrogenase as simple model to engineer nitrogen fixation in yeast and plant mitochondria.
在真核生物中工程化固氮酶受到其遗传复杂性和蛋白成分对氧气敏感性的阻碍。在三种类型的固氮酶中，Fe 只有固氮酶被认为是最简单的一种，因为它的功能依赖于比同源且更复杂的 Mo 和 V 固氮酶更少的基因产物。在这里，我们展示了稳定的 Fe 只有固氮酶成分蛋白在低氧酵母线粒体基质中的表达。分离的 Fe 蛋白（AnfH）在电子供体向 NifDK 的传递中具有活性，将乙炔还原为乙烯。辅助蛋白 NifU、NifS 和 NifM 对于 Fe 蛋白的功能不是必需的。FeFe 蛋白以 apo-AnfDK 复合物的形式存在，AnfG 亚基要么松散结合，要么完全无法与之相互作用。Apo-AnfDK 可以通过简单地添加 FeMo-co 在体外被激活用于乙炔还原，这表明即使没有共表达的 NifU 和 NifS，P 簇也预先存在。这项工作加强了使用 Fe 只有固氮酶作为工程化酵母和植物线粒体固氮的简单模型。
