AUTHOR=Burén Stefan , Jiang Xi , López-Torrejón Gema , Echavarri-Erasun Carlos , Rubio Luis M. 

TITLE=Purification and In Vitro Activity of Mitochondria Targeted Nitrogenase Cofactor Maturase NifB

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.01567

DOI=10.3389/fpls.2017.01567

ISSN=1664-462X

ABSTRACT=<p>Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O<sub>2</sub>-sensitive <italic>S</italic>-adenosyl methionine (SAM)–radical enzyme that provides the key metal cluster intermediate (NifB-co) for the biosyntheses of the active-site cofactors of all three types of nitrogenases. NifB and NifB-co are unique to diazotrophic organisms. In this work, we have expressed synthetic codon-optimized versions of NifB from the γ-proteobacterium <italic>Azotobacter vinelandii</italic> and the thermophilic methanogen <italic>Methanocaldococcus infernus</italic> in <italic>Saccharomyces cerevisiae</italic> and in <italic>Nicotiana benthamiana</italic>. NifB proteins were targeted to the mitochondria, where O<sub>2</sub> consumption is high and bacterial-like [Fe-S] cluster assembly operates. In yeast, NifB proteins were co-expressed with NifU, NifS, and FdxN proteins that are involved in NifB [Fe–S] cluster assembly and activity. The synthetic version of thermophilic NifB accumulated in soluble form within the yeast cell, while the <italic>A. vinelandii</italic> version appeared to form aggregates. Similarly, NifB from <italic>M. infernus</italic> was expressed at higher levels in leaves of <italic>Nicotiana benthamiana</italic> and accumulated as a soluble protein while <italic>A. vinelandii</italic> NifB was mainly associated with the non-soluble cell fraction. Soluble <italic>M. infernus</italic> NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the <italic>in vitro</italic> FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening <italic>nif</italic> genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase.</p>