Cophenetic correlation analysis as a strategy to select phylogenetically informative proteins: an example from the fungal kingdom

<p>Abstract</p> <p>Background</p> <p>The construction of robust and well resolved phylogenetic trees is important for our understanding of many, if not all biological processes, including speciation and origin of higher taxa, genome evolution, metabolic diversification, multicellularity, origin of life styles, pathogenicity and so on. Many older phylogenies were not well supported due to insufficient phylogenetic signal present in the single or few genes used in phylogenetic reconstructions. Importantly, single gene phylogenies were not always found to be congruent. The phylogenetic signal may, therefore, be increased by enlarging the number of genes included in phylogenetic studies. Unfortunately, concatenation of many genes does not take into consideration the evolutionary history of each individual gene. Here, we describe an approach to select informative phylogenetic proteins to be used in the Tree of Life (TOL) and barcoding projects by comparing the cophenetic correlation coefficients (CCC) among individual protein distance matrices of proteins, using the fungi as an example. The method demonstrated that the quality and number of concatenated proteins is important for a reliable estimation of TOL. Approximately 40–45 concatenated proteins seem needed to resolve fungal TOL.</p> <p>Results</p> <p>In total 4852 orthologous proteins (KOGs) were assigned among 33 fungal genomes from the Asco- and Basidiomycota and 70 of these represented single copy proteins. The individual protein distance matrices based on 531 concatenated proteins that has been used for phylogeny reconstruction before <abbrgrp><abbr bid="B14">14</abbr></abbrgrp> were compared one with another in order to select those with the highest CCC, which then was used as a reference. This reference distance matrix was compared with those of the 70 single copy proteins selected and their CCC values were calculated. Sixty four KOGs showed a CCC above 0.50 and these were further considered for their phylogenetic potential. Proteins belonging to the cellular processes and signaling KOG category seem more informative than those belonging to the other three categories: information storage and processing; metabolism; and the poorly characterized category. After concatenation of 40 proteins the topology of the phylogenetic tree remained stable, but after concatenation of 60 or more proteins the bootstrap support values of some branches decreased, most likely due to the inclusion of proteins with lowers CCC values. The selection of protein sequences to be used in various TOL projects remains a critical and important process. The method described in this paper will contribute to a more objective selection of phylogenetically informative protein sequences.</p> <p>Conclusion</p> <p>This study provides candidate protein sequences to be considered as phylogenetic markers in different branches of fungal TOL. The selection procedure described here will be useful to select informative protein sequences to resolve branches of TOL that contain few or no species with completely sequenced genomes. The robust phylogenetic trees resulting from this method may contribute to our understanding of organismal diversification processes. The method proposed can be extended easily to other branches of TOL.</p

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 Azotobacter vinelandii. We show here that three proteins, NafH, NifW, and NifZ, copurify with MoFe protein produced by an A. vinelandii strain deficient in both FeMo-cofactor formation and P-cluster maturation. In contrast, two different proteins, NifY and NafY, copurified with MoFe protein deficient only in FeMo-cofactor formation. We refer to proteins associated with immature MoFe protein in the following as “assembly factors.” Copurifications of such assembly factors with MoFe protein produced in different genetic backgrounds revealed their sequential and differential interactions with MoFe protein during the maturation process. We found that these interactions occur in the order NafH, NifW, NifZ, and NafY/NifY. Interactions of NafH, NifW, and NifZ with immature forms of MoFe protein preceded completion of P-cluster maturation, whereas interaction of NafY/NifY preceded FeMo-cofactor insertion. Because each assembly factor could independently bind an immature form of MoFe protein, we propose that subpopulations of MoFe protein–assembly factor complexes represent MoFe protein captured at different stages of a sequential maturation process. This suggestion was supported by separate isolation of three such complexes, MoFe protein–NafY, MoFe protein–NifY, and MoFe protein–NifW. We conclude that factors involved in MoFe protein maturation sequentially bind and dissociate in a dynamic process involving several MoFe protein conformational states

Purification and characterization of NifB from Chloroflexi

NifB tiene un papel crucial en la biogénesis de la nitrogenasa, enzima responsable de la fijación del nitrógeno atmosférico (N2) a amonio (NH3), proceso conocido como Fijación Biológica del Nitrógeno. NifB es una proteína que pertenece a una familia de proteínas conocida como ?SAM-radical proteins? y cataliza la síntesis del cofactor metálico NifB-co, [Fe8-S9-C] a partir de dos ?clusters? sulfo-férricos del tipo [Fe4-S4] y dos moléculas de SAM. NifB-co sirve como intermediario en la biosíntesis de los sitios activos (cofactores metálicos) de todas las nitrogenasas conocidas, donde la más común es la nitrogenasa de molibdeno, cuyo cofactor, conocido como FeMo-co, consiste en [Fe7-S9-C-Mo-Homocitrato]. Inicialmente, NifB fue purificado a partir de microorganismos diazotrofos como Azotobacter vinelandii o Klebsiella oxytoca. Ambas especies son ?-proteobacterias mesofílicas de vida libre, distribuidas en suelos cuyo NifB presenta una arquitectura con dos dominios, el dominio SAM-radical y el dominio NifX. Sin embargo, en estudios recientes se ha purificado satisfactoriamente NifBs procedentes de metanógenos termofílicos que presentan únicamente el dominio SAM-radical en su estructura, expresados de forma heteróloga en E. coli. En el trabajo presentado en el congreso ENFC 2018, se ha purificado y caracterizado NifB de una bacteria perteneciente al filo Chloroflexi, Roseiflexus sp. RS.1. Este NifB termorresistente, es estructuralmente similar a NifBs procedentes de arqueas y únicamente presenta el dominio SAM. En este estudio se muestras las propiedades bioquímicas de NifB de Roseiflexus y su capacidad para sintetizar NifB-co en ensayos de síntesis e inserción de FeMo-co in vitro, para la activación de la enzima nitrogenasa. NifB de Roseiflexus es termorresistente, llega a alcanzar un total de 9 átomos de hierro por monómero y presenta propiedades espectroscópicas compatibles con la presencia de tres clusters [Fe4-S4] tal y como se ha reportado para su homólogo NifB de Methanocalcococcus infernus. ----------ABSTRACT---------- NifB has a crucial role in the biogenesis of active nitrogenase, the enzyme responsible of fixing atmospheric N2 to NH3 in a process known as biological nitrogen fixation (BNF). NifB is a SAM-radical protein that catalyzes the synthesis of a metal cluster, NifB-co [Fe8-S9-C], from two [Fe4-S4] cluster units and a molecule of SAM. NifB-co serves as intermediate in the biosynthesis of the active-site cofactors of all known nitrogenases(1,2). NifB has been purified from mesophilicγ–proteobacteria, having dual domain architectures based on a SAM-radical domain and a NifX-like domain(3). However, recent studies have successfully used NifB proteins from thermophillic methanogens that present a SAM-radical-domain-only architecture(4,5). In this work, NifB from Roseiflexus sp. RS-1 was heterologously expressed in Escherichia coli and purified to be used as adiitional model to further understand how NifB proteins synthesize NifB-co. Roseiflexus sp. RS-1 is a filamentous bacterium that belongs to the phylum Chloroflexi. It has thermophilic lifestyle, growing at 60ºC. This phylum is found near the most primitive branches of the phylogenetic tree, constituting an interesting candidate to study the most primitive forms of NifB and its co-evolution with the nitrogenase complex

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. Here we study the diversity and phylogeny of naturally occurring NifB proteins, their protein architecture and the functions of the distinct NifB domains in order to understand what defines a catalytically active NifB. Focus is on NifB from the thermophile Chlorobium tepidum (two-domain architecture), the hyperthermophile Methanocaldococcus infernus (single-domain architecture) and the mesophile Klebsiella oxytoca (two-domain architecture), showing in silico characterization of their nitrogen fixation (nif) gene clusters, conserved NifB motifs, and functionality. C. tepidum and M. infernus NifB were able to complement an Azotobacter vinelandii (ΔnifB) mutant restoring the Nif+ phenotype and thus demonstrating their functionality in vivo. In addition, purified C. tepidum NifB exhibited activity in the in vitro NifB-dependent nitrogenase reconstitution assay. Intriguingly, changing the two-domain K. oxytoca NifB to single-domain by removal of the C-terminal NifX-like extension resulted in higher in vivo nitrogenase activity, demonstrating that this domain is not required for nitrogen fixation in mesophiles

Electron Paramagnetic Resonance Characterization of Three Iron–Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from Methanocaldococcus infernus

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe-S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron-molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5'-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe-4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed

Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae

ABSTRACTBiological nitrogen fixation, the conversion of inert N2 to metabolically tractable NH3, is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo-R-homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae, represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae, targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii. However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae. Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism.IMPORTANCEBiological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo-R-homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae. NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae. FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms

Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana

13 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP)Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 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. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.This study was supported, in whole or in part, by the Bill & Melinda Gates Foundation (OPP1143172 and INV 005889). Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 Generic License has already been assigned to the Author Accepted Manuscript version that might arise from this submission. X.J. was supported by a doctoral fellowship from Universidad Politécnica de MadridPeer reviewe

Structural insights into the mechanism of the radical SAM carbide synthase NifB, a key nitrogenase cofactor maturating enzyme

International audienceNitrogenase 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 [MoFe7S9C-(R)-homocitrate] species designated FeMo-cofactor, whose biosynthesis and insertion requires the action of over a dozen maturation proteins provided by the NIF (for NItrogen Fixation) assembly machinery. Among them, the radical SAM protein NifB plays an essential role, concomitantly inserting a carbide ion and coupling two [Fe4S4] clusters to form a [Fe8S9C] precursor called NifB-co. Here we report on the X-ray structure of NifB from Methanotrix thermoacetophila at 1.95 Å resolution in a state pending the binding of one [Fe4S4] cluster substrate. The overall NifB architecture indicates that this enzyme has a single SAM binding site, which at this stage is occupied by cysteine residue 62. The structure reveals a unique ligand binding mode for the K1 cluster involving cysteine residues 29 and 128 in addition to histidine 42 and glutamate 65. The latter, together with cysteine 62, belongs to a loop inserted in the active site, likely protecting the already present [Fe4S4] clusters. These two residues regulate the sequence of events, controlling SAM dual reactivity and preventing unwanted radical-based chemistry before the K2 [Fe4S4] cluster substrate is loaded into the protein. The location of K1 cluster, too far away from the SAM binding site, supports a mechanism in which the K2 cluster is the site of methylation

Electron Paramagnetic Resonance Characterization of Three Iron–Sulfur Clusters Present in the Nitrogenase Cofactor Maturase NifB from <i>Methanocaldococcus infernus</i>

NifB utilizes two equivalents of <i>S</i>-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe–S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron–molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen <i>Methanocaldococcus infernus</i> is a radical SAM enzyme able to reductively cleave SAM to 5′-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe–4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed