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Descripción
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| The radical S-adenosylmethionine (SAM) enzyme NifB occupies a cen- tral and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which consti- tutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Saccharo- myces cerevisiae. Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different ex- pression levels of mitochondria-targeted nif genes (nifUSXB and fdxN) were integrated into the chromosome. Two combinatorial li- braries totaling 0.7 Mb were constructed: An expression library of 6 partial clusters, including nifUSX and fdxN, and a library consisting of 28 different nifB genes mined from the Structure?Function Link- age Database and expressed at different levels according to a facto- rial design. We show that coexpression in yeast of the nitrogenase maturation proteins NifU, NifS, and FdxN from Azotobacter vinelandii with NifB from the archaea Methanocaldococcus infernus or Meth- anothermobacter thermautotrophicus yields NifB proteins equipped with [Fe-S] clusters that, as purified, support in vitro formation of NifB-co. Proof of in vivo NifB-co formation was additionally obtained. NifX as purified from aerobically cultured S. cerevisiae coexpressing M. thermautotrophicus NifB with A. vinelandii NifU, NifS, and FdxN, and engineered yeast SAM synthase supported FeMo-co synthesis, indicative of NifX carrying in vivo-formed NifB-co. This study defines the minimal genetic determinants for the formation of the key precur- sor in the nitrogenase cofactor biosynthetic pathway in a eukaryotic organism. | |
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Internacional
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JCR del ISI
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Si |
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Título de la revista
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Proceedings of the National Academy of Sciences of the United States of America |
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ISSN
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0027-8424 |
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Factor de impacto JCR
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9,504 |
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Información de impacto
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Datos JCR del año 2017 |
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Volumen
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116 |
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DOI
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10.1073/pnas.1904903116 |
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Número de revista
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50 |
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Desde la página
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1882 |
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Hasta la página
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1896 |
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Mes
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NOVIEMBRE |
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Ranking
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