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

Active NifB is a milestone in the process of engineering nitrogen fixing plants. NifB is an extremely O2-sensitive S-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 ?-proteobacterium Azotobacter vinelandii and the thermophilic methanogen Methanocaldococcus infernus in Saccharomyces cerevisiae and in Nicotiana benthamiana. NifB proteins were targeted to the mitochondria, where O2 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 A. vinelandii version appeared to form aggregates. Similarly, NifB from M. infernus was expressed at higher levels in leaves of Nicotiana benthamiana and accumulated as a soluble protein while A. vinelandii NifB was mainly associated with the non-soluble cell fraction. Soluble M. infernus NifB was purified from aerobically grown yeast and biochemically characterized. The purified protein was functional in the in vitro FeMo-co synthesis assay. This work presents the first active NifB protein purified from a eukaryotic cell, and highlights the importance of screening nif genes from different organisms in order to sort the best candidates to assemble a functional plant nitrogenase

Fluorescent in vivo imaging of reactive oxygen species and redox potential in plants

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networksThis work was funded by a grant from the Spanish Ministry of Economy and Competitiveness ( AGL2014–53771-R ). Alfonso Blázquez-Castro acknowledges funding under the Marie Skłodowska-Curie Action COFUND 2015 (EU project 713366 – InterTalentum

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. The production of NifB in plants is challenging because this protein is often insoluble in eukaryotic cells, and its [Fe−S] clusters are extremely unstable and sensitive to O2. As a first step to address this challenge, we generated transgenic rice plants expressing NifB from the Archaea Methanocaldococcus infernus and Methanothermobacter thermautotrophicus. The recombinant proteins were targeted to the mitochondria to limit exposure to O2 and to have access to essential [4Fe−4S] clusters required for NifB-co biosynthesis. M. infernus and M. thermautotrophicus NifB accumulated as soluble proteins in planta, and the purified proteins were functional in the in vitro FeMo-co synthesis assay. We thus report NifB protein expression and purification from an engineered staple crop, representing a first step in the biosynthesis of a functional NifDK complex, as required for independent biological nitrogen fixation in cereals

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 N2. 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 O2 sensitive and only sparingly soluble in non-native hosts. We generated transgenic rice plants expressing the nitrogenase structural component, Fe protein (NifH), which carries a [4Fe-4S] cluster in its active form. NifH from Hydrogenobacter thermophilus was targeted to mitochondria together with the putative peptidyl prolyl cis‐trans isomerase NifM from Azotobacter vinelandii to assist in NifH polypeptide folding. The isolated NifH was partially active in electron transfer to the MoFe protein nitrogenase component (NifDK) and in the biosynthesis of the nitrogenase iron-molybdenum cofactor (FeMo-co), two fundamental roles for NifH in N2 fixation. NifH functionality was, however, limited by poor [4Fe-4S] cluster occupancy, highlighting the importance of in vivo [Fe-S] cluster insertion and stability to achieve biological N2 fixation in planta. Nevertheless, the expression and activity of a nitrogenase component in rice plants represents the first major step to engineer functional nitrogenase in cereal crops

Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

Background: The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

Transport och funktion av CAH1 : Karakterisering av en ny transportväg för proteiner till växtcellens kloroplast

The chloroplast is the organelle within a plant cell where photosynthesis takes place. This organelle originates from a cyanobacterium that was engulfed by a eukaryotic cell. During the transition from endosymbiont to organelle most of the cyanobacterial genes were transferred to the nuclear genome of the host cell, resulting in a chloroplast with a much reduced genome that requires massive import of gene products (proteins) back to the organelle. The majority of these proteins are translated in the cytosol as pre-proteins containing targeting information that directs them to a translocon complex in the chloroplast envelope, the Toc-Tic system, through which these proteins are transported. We have identified a protein in the model plant Arabidopsis thaliana, CAH1, that is trafficked via the endomembrane system (ER/Golgi apparatus) to the chloroplast instead of using the Toc-Tic machinery. This transport is partly mediated by canonical vesicle trafficking elements involved in ER to Golgi transport, such as Sar1 and RabD GTPases. Analysis of point mutated variants of CAH1 showed that both N-linked glycans and an intra-molecular disulphide bridge are required for correct folding, trafficking and function of the protein. Since chloroplasts lack N-glycosylation machinery, we propose that a route for chloroplast proteins that require endomembrane-specific post-translational modifications for their functionality exists as a complement to the Toc-Tic system. We also show that mutant plants with disrupted CAH1 gene expression have reduced rates of CO2 uptake and accumulate lower amounts of starch compared to wild-type plants, indicating an important function of the CAH1 protein for the photosynthetic capacity of Arabidopsis. Further study of CAH1 will not only be important to reveal its role in photosynthesis, but characterization of this novel protein pathway to the chloroplast can also shed light on how the plant cell evolved and clarify the purpose of keeping several chloroplast import pathways working in parallel. In addition, knowledge about this pathway could increase the opportunities for using plants as bio-factories for production of recombinant glycoproteins, which make up the vast majority of the bio-pharmaceutical molecules.Kloroplasten är den organell i växtcellen där fotosyntesen sker. Denna organell härstammar från en cyanobakterie som togs upp av en eukaryot cell. Under omvandlingen från endosymbiont till organell har de flesta av den ursprungliga cyanobakteriens gener flyttats över till växtcellens eget kärngenom, vilket resulterat i en kloroplast som endast kan producera ett fåtal av de proteiner den behöver och som istället kräver att en mängd genprodukter (proteiner) transporteras tillbaka till organellen. De flesta av dessa proteiner syntetiseras i cytosolen som polypeptider innehållande en speciell signal för kloroplasten, och tranporteras över kloroplastens dubbelmembran (envelop) med hjälp av ett specifikt importsystem (Toc-Tic). Vi har identifierat ett protein i modellväxten Arabidopsis thaliana (CAH1) som istället för att använda Toc-Tic tranporteras via det endomembrana systemet (ER/Golgi). Transporten sker delvis med hjälp av faktorer involverade i normal vesikeltransport, t.ex. Sar1 och RabD GTPaser (mellan ER och Golgi). Genom att uttycka och analysera punktmuterade varianter av CAH1 har vi kunnat visa att både sockergrupper kopplade till proteinet, samt en intern svavelbrygga, är nödvändiga för korrekt veckning, transport och funktion av proteinet. Då kloroplasten saknar eget maskineri för att koppla sådana sockergrupper till proteiner så föreslår vi att anledningen till att denna rutt existerar, som ett komplement till Toc-Tic, är för att proteiner beroende av denna typ av modifiering ska kunna finnas i kloroplasten. Vi visar också att muterade växter som inte kan uttrycka genen som kodar för CAH1 uppvisar lägre upptag av CO2, samt ackumulerar mindre stärkelse än vildtypplantor, vilket antyder att CAH1 har en viktig funktion för den fotosyntetiska förmågan hos Arabidopsis. För att kunna fastställa den exakta funktionen för CAH1 kommer ytterliga studier att vara nödvändiga. En fördjupad karaktärisering av transportvägen som CAH1 följer till kloroplasten kan dessutom ge kunskap om hur växtcellen uppkom, samt besvara varför flera importvägar arbetar till synes parallellt med varandra. Kunskap om denna transportväg kan även bidra med användbar information i försöken att nyttja växter till att uttrycka rekombinanta N-glykosylerade proteiner, t. ex. antikroppar och vacciner

In vivo ROS and redox potential fluorescent detection in plants: Present approaches and future perspectives

Reactive oxygen species (ROS) are metabolic by-products in aerobic organisms including plants. Endogenously produced ROS act as cellular messengers and redox regulators involved in several plant biological processes, but excessive accumulation of ROS cause oxidative stress and cell damage. Understanding ROS signalling and stress responses requires precise imaging and quantification of local, subcellular and global ROS dynamics with high selectivity, sensitivity, and spatiotemporal resolution. Several fluorescent vital dyes have been tested so far, which helped to provide relevant spatially resolved information of oxidative stress dynamics in plants subjected to harmful environmental conditions. However, certain plant characteristics, such as high background fluorescence of plant tissues in vivo and antioxidant mechanisms, can interfere with ROS detection. The development of improved small-molecule fluorescent dyes and protein-based ROS sensors targeted to subcellular compartments will enable in vivo monitoring of ROS and redox changes in photosynthetic organismsThis work was funded by a grant from the Spanish Ministry of Economy and Competitiveness (AGL2014-53771-R

Biosynthesis of Nitrogenase Cofactors

48 Pág.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-R-homocitrate] group called FeMo-co. Formation of nitrogenase metalloclusters requires the participation of the structural nitrogenase components and many accessory proteins, and occurs both in situ, for the P-cluster, and in external assembly sites for FeMo-co. The biosynthesis of FeMo-co is performed stepwise and involves molecular scaffolds, metallochaperones, radical chemistry, and novel and unique biosynthetic intermediates. This review provides a critical overview of discoveries on nitrogenase cofactor structure, function, and activity over the last four decades.This work has been funded by the Bill & Melinda Gates Foundation Grant No. OPP1143172 and by FEDER / Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación / Proyecto 2017-88475-R.Peer reviewe

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

11 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP)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.This work was supported by the Bill and Melinda Gates Foundation Grant OPP1143172 to LMR.Peer reviewe