Iron-molybdenum cofactor synthesis by a thermophilic nitrogenase devoid of the scaffold NifEN.

The maturation and installation of the active site metal cluster (FeMo-co, FeSCMo--homocitrate) in Mo-dependent nitrogenase requires the protein product of the gene for production of the FeS cluster precursor (NifB-co, [FeSC]) and the action of the maturase complex composed of the protein products from the and genes. However, some putative diazotrophic bacteria, like sp. RS-1, lack the genes, suggesting an alternative pathway for maturation of FeMo-co that does not require NifEN.

Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of .

Biological nitrogen fixation, the conversion of inert N to metabolically tractable NH, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo--homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as , represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation.

Iron Homeostasis in .

Iron is an essential nutrient for all life forms. Specialized mechanisms exist in bacteria to ensure iron uptake and its delivery to key enzymes within the cell, while preventing toxicity. Iron uptake and exchange networks must adapt to the different environmental conditions, particularly those that require the biosynthesis of multiple iron proteins, such as nitrogen fixation.

Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in .

Understanding how Nature accomplishes the reduction of inert nitrogen gas to form metabolically tractable ammonia at ambient temperature and pressure has challenged scientists for more than a century. Such an understanding is a key aspect toward accomplishing the transfer of the genetic determinants of biological nitrogen fixation to crop plants as well as for the development of improved synthetic catalysts based on the biological mechanism. Over the past 30 years, the free-living nitrogen-fixing bacterium emerged as a preferred model organism for mechanistic, structural, genetic, and physiological studies aimed at understanding biological nitrogen fixation.

Functional expression of the nitrogenase Fe protein in transgenic rice.

Engineering cereals to express functional nitrogenase is a long-term goal of plant biotechnology and would permit partial or total replacement of synthetic N fertilizers by metabolization of atmospheric N. Developing this technology is hindered by the genetic and biochemical complexity of nitrogenase biosynthesis. Nitrogenase and many of the accessory proteins involved in its assembly and function are O sensitive and only sparingly soluble in non-native hosts.

A directed genome evolution method to enhance hydrogen production in .

Nitrogenase-dependent H production by photosynthetic bacteria, such as , has been extensively investigated. An important limitation to increase H production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered strains that emitted fluorescence in response to H and used them to identify mutations in the nitrogenase Fe protein leading to H overproduction.

Nitrogenase Cofactor Maturase NifB Isolated from Transgenic Rice is Active in FeMo-co Synthesis.

The engineering of nitrogen fixation in plants requires assembly of an active prokaryotic nitrogenase complex, which is yet to be achieved. Nitrogenase biogenesis relies on NifB, which catalyzes the formation of the [8Fe-9S-C] metal cluster NifB-co. This is the first committed step in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) found at the nitrogenase active site.

A colorimetric method to measure in vitro nitrogenase functionality for engineering nitrogen fixation.

Biological nitrogen fixation (BNF) is the reduction of N into NH in a group of prokaryotes by an extremely O-sensitive protein complex called nitrogenase. Transfer of the BNF pathway directly into plants, rather than by association with microorganisms, could generate crops that are less dependent on synthetic nitrogen fertilizers and increase agricultural productivity and sustainability. In the laboratory, nitrogenase activity is commonly determined by measuring ethylene produced from the nitrogenase-dependent reduction of acetylene (ARA) using a gas chromatograph.

Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of .

Engineering plants to synthesize nitrogenase and assimilate atmospheric N will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors.

Environment and coordination of FeMo-co in the nitrogenase metallochaperone NafY.

In nitrogenase biosynthesis, the iron-molybdenum cofactor (FeMo-co) is externally assembled at scaffold proteins and delivered to the NifDK nitrogenase component by the NafY metallochaperone. Here we have used nuclear magnetic resonance, molecular dynamics, and functional analysis to elucidate the environment and coordination of FeMo-co in NafY. H stands as the key FeMo-co ligand.

An unexpected P-cluster like intermediate to the nitrogenase FeMo-co.

The nitrogenase MoFe protein contains two different FeS centers, the P-cluster and the iron-molybdenum cofactor (FeMo-co). The former is a [FeS] center responsible for conveying electrons to the latter, a [MoFeSC-()-homocitrate] species, where N reduction takes place. NifB is arguably the key enzyme in FeMo-co assembly as it catalyzes the fusion of two [FeS] clusters and the insertion of carbide and sulfide ions to build NifB-co, a [FeSC] precursor to FeMo-co.

Biosynthesis of cofactor-activatable iron-only nitrogenase in Saccharomyces cerevisiae.

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.

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N-fixation in plants.

Engineering nitrogen fixation in eukaryotes requires high expression of functional nitrogenase structural proteins, a goal that has not yet been achieved. Here we build a knowledge-based library containing 32 nitrogenase nifH sequences from prokaryotes of diverse ecological niches and metabolic features and combine with rapid screening in tobacco to identify superior NifH variants for plant mitochondria expression. Three NifH variants outperform in tobacco mitochondria and are further tested in yeast.

Transit Peptides From Photosynthesis-Related Proteins Mediate Import of a Marker Protein Into Different Plastid Types and Within Different Species.

Nucleus-encoded plastid proteins are synthesized as precursors with N-terminal targeting signals called transit peptides (TPs), which mediate interactions with the translocon complexes at the outer (TOC) and inner (TIC) plastid membranes. These complexes exist in multiple isoforms in higher plants and show differential specificity and tissue abundance. While some show specificity for photosynthesis-related precursor proteins, others distinctly recognize nonphotosynthetic and housekeeping precursor proteins.

Structural Insights into the Mechanism of the Radical SAM Carbide Synthase NifB, a Key Nitrogenase Cofactor Maturating Enzyme.

Nitrogenase is a key player in the global nitrogen cycle, as it catalyzes the reduction of dinitrogen into ammonia. The active site of the nitrogenase MoFe protein corresponds to a [MoFeSC-()-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for trogen ixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [FeS] clusters to form a [FeSC] precursor called NifB-co.

Use of synthetic biology tools to optimize the production of active nitrogenase Fe protein in chloroplasts of tobacco leaf cells.

The generation of nitrogen fixing crops is considered a challenge that could lead to a new agricultural 'green' revolution. Here, we report the use of synthetic biology tools to achieve and optimize the production of active nitrogenase Fe protein (NifH) in the chloroplasts of tobacco plants. Azotobacter vinelandii nitrogen fixation genes, nifH, M, U and S, were re-designed for protein accumulation in tobacco cells.

Biosynthesis of Nitrogenase Cofactors.

Nitrogenase harbors three distinct metal prosthetic groups that are required for its activity. The simplest one is a [4Fe-4S] cluster located at the Fe protein nitrogenase component. The MoFe protein component carries an [8Fe-7S] group called P-cluster and a [7Fe-9S-C-Mo--homocitrate] group called FeMo-co.

Biosynthesis of the nitrogenase active-site cofactor precursor NifB-co in .

The radical -adenosylmethionine (SAM) enzyme NifB occupies a central and essential position in nitrogenase biogenesis. NifB catalyzes the formation of an [8Fe-9S-C] cluster, called NifB-co, which constitutes the core of the active-site cofactors for all 3 nitrogenase types. Here, we produce functional NifB in aerobically cultured Combinatorial pathway design was employed to construct 62 strains in which transcription units driving different expression levels of mitochondria-targeted genes ( and ) were integrated into the chromosome.

Genetic and Biochemical Analysis of the Molybdenum Storage Protein.

The N fixing bacterium carries a molybdenum storage protein, referred to as MoSto, able to bind 25-fold more Mo than needed for maximum activity of its Mo nitrogenase. Here we have investigated a plausible role of MoSto as obligate intermediate in the pathway that provides Mo for the biosynthesis of nitrogenase iron-molybdenum cofactor (FeMo-co). The FeMo-co synthesis and insertion assay demonstrated that purified MoSto functions as Mo donor and that direct interaction with FeMo-co biosynthetic proteins stimulated Mo donation.

Sequential and differential interaction of assembly factors during nitrogenase MoFe protein maturation.

Nitrogenases reduce atmospheric nitrogen, yielding the basic inorganic molecule ammonia. The nitrogenase MoFe protein contains two cofactors, a [7Fe-9S-Mo-C-homocitrate] active-site species, designated FeMo-cofactor, and a [8Fe-7S] electron-transfer mediator called P-cluster. Both cofactors are essential for molybdenum-dependent nitrogenase catalysis in the nitrogen-fixing bacterium We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an strain deficient in both FeMo-cofactor formation and P-cluster maturation.

Diversity and Functional Analysis of the FeMo-Cofactor Maturase NifB.

One of the main hurdles to engineer nitrogenase in a non-diazotrophic host is achieving NifB activity. NifB is an extremely unstable and oxygen sensitive protein that catalyzes a low-potential SAM-radical dependent reaction. The product of NifB activity is called NifB-co, a complex [8Fe-9S-C] cluster that serves as obligate intermediate in the biosyntheses of the active-site cofactors of all known nitrogenases.

State of the art in eukaryotic nitrogenase engineering.

Improving the ability of plants and plant-associated organisms to fix and assimilate atmospheric nitrogen has inspired plant biotechnologists for decades, not only to alleviate negative effects on nature from increased use and availability of reactive nitrogen, but also because of apparent economic benefits and opportunities. The combination of recent advances in synthetic biology and increased knowledge about the biochemistry and biosynthesis of the nitrogenase enzyme has made the seemingly remote and for long unreachable dream more possible. In this review, we will discuss strategies how this could be accomplished using biotechnology, with a special focus on recent progress on engineering plants to express its own nitrogenase.

Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria.

Background: There is a need for the development of synthetic biology methods and tools to facilitate rapid and efficient engineering of yeast that accommodates the needs of specific biotechnology projects. In particular, the manipulation of the mitochondrial proteome has interesting potential applications due to its compartmentalized nature. One of these advantages resides in the fact that metalation occurs after protein import into mitochondria, which contains pools of iron, zinc, copper and manganese ions that can be utilized in recombinant metalloprotein metalation reactions.