Non-canonical regulation of glutathione and trehalose biosynthesis characterizes non-Saccharomyces wine yeasts with poor performance in active dry yeast production

Several yeast species, belonging to Saccharomyces and non-Saccharomyces genera, play fundamental roles during spontaneous must grape fermentation, and recent studies have shown that mixed fermenta-tions, co-inoculated with S. cerevisiae and non-Saccharomyces strains, can improve wine organoleptic properties. During active dry yeast (ADY) produc-tion, antioxidant systems play an essential role in yeast survival and vitality as both biomass propagation and dehydration cause cellular oxidative stress and negatively affect technological performance. Mechanisms for adaptation and resistance to desiccation have been described for S. cerevisiae, but no data are available on the physiology and oxidative stress response of non-Saccharomyces wine yeasts and their potential impact on ADY production. In this study we analyzed the oxidative stress response in several non-Saccharomyces yeast species by measuring the activity of reactive oxygen species (ROS) scavenging enzymes, e.g., catalase and glutathione reductase, accumulation of protective metabolites, e.g., trehalose and reduced glutathi-one (GSH), and lipid and protein oxidation levels. Our data suggest that non-canonical regulation of glutathione and trehalose biosynthesis could cause poor fermentative performance after ADY production, as it corroborates the corrective effect of antioxidant treatments, during biomass propagation, with both pure chemicals and food-grade argan oil

Improvement of the Activity of a Fungal Versatile-Lipase Toward Triglycerides: An in silico Mechanistic Description

Some enzymes that belong to the Candida rugosa-like lipase family (abH03. 01) combine the activities of lipases and sterol esterases. Thus, they can act on water-insoluble carboxylic esters releasing long-chain fatty acids but also on sterol esters, although with different activity and affinity. The differences in the catalytic properties among the proteins of this family are explained by small changes in the hydrophobicity of some regions. One of such versatile enzymes is the sterol esterase/lipase from Ophiostoma piceae (OPE) that acts very efficiently on the two types of substrates. Structurally, OPE is characterized by the presence of a lid formed by a α-helix and two 310-helices rich in hydrophobic amino acids. In this study, the ope gene was modified by directed mutagenesis in order to change specific amino acids in the lid region to modify its structure with the aim of increasing its hydrophobicity. Several recombinant forms of OPE were heterologously produced in Pichia pastoris. In silico molecular dynamics simulations have been used to decipher the mechanistic principles behind the improvements in substrate catalysis. The analyses suggested that the enhanced activity toward hydrophobic substrates such as triglycerides could be due to a better stabilization of the substrate in the lid region as a result of an increased hydrophobicity and an improved topology. These results indicate that in silico simulations can be useful for the optimization of the activity of lipases from the C. rugose-like family for different biotechnological applications

Insights into nitrogenase biosynthesis obtained from thermophilic prokaryotes

Biological nitrogen fixation (BNF) is a high-energy cost enzymatic process that reductively breaks the triple bond of nitrogen gas (N2; N≡N) to render two ammonia (NH3) molecules. BNF is carried out, exclusively, by a few microorganisms belonging to the Bacteria and Archaea domains, known as diazotrophs. All diazotrophs fix N2 using a two-protein component nitrogenase enzyme. Based on its composition of metal cofactors, which are essential to activity, nitrogenases are classified as: molybdenum (Mo), vanadium (V) or iron-only (Fe) nitrogenases. All diazotrophs contain at least a Mo-nitrogenase, while some diazotrophs additionally contain V or Fe- nitrogenases. Therefore, the Mo-nitrogenase is the most abundant and is suggested to be the evolutionary predecessor of V- and Fe-nitrogenases. Mo-nitrogenase enzyme, as well as proteins required for its assembly, function, and regulation, are encoded in nitrogen fixation (nif) genes. The quantity and composition of a nif gene complement varies depending on the physiology and ecology of each diazotroph. However, a minimal set of six nif genes (nifHDKENB) has been established as essential criterium for being able to perform Mo-dependent N2 fixation. The present thesis investigates nitrogenase from the thermophilic bacterium Roseiflexus sp. RS-1, which appears depend only on nifHBDK. The NifH and NifDK nitrogenase structural components, and the biosynthetic protein NifB, have been characterized. Demonstration of in vitro activity present in purified Roseiflexus sp. nitrogenase components shows the existence of an alternative pathway for the biosynthesis of its active-site iron-molybdenum cofactor (FeMo-co). In Roseiflexus sp. FeMo-co biosynthesis seems to be NifEN-independent while NifDK has dual capability as FeMo-co maturase and nitrogenase enzyme. These results suggest that Roseiflexus sp. carries an enzyme complex likely resembling the predecessor of current Mo-nitrogenases before the events of duplication and divergence of nifDK and nifEN genes. Additionally, this thesis includes the first X-ray atomic structure resolution of a NifB protein. Because the Roseiflexus sp. NifB protein could not be crystallized, structure of the homologous NifB from Methanotrix thermoacetophila was solved in collaboration with the group of Dr. Yvain Nicolet at the Institute de Biologie Structurale. A novel [Fe4S4] cluster coordination involving two cysteine, one histidine and one glutamate residues was observed. NifB site directed mutagenesis and biochemical analyses performed as part of this thesis allowed us to refine a catalytic model for NifB-co synthesis, where a loop constituted by residues C62 to E65 has a central role in the orchestration of SAM binding/cleavage and [4Fe-4S] cluster stabilization. ----------RESUMEN---------- La fijación biológica de nitrógeno (FBN) es un proceso enzimático con un alto coste energético encargado de la rotura del triple enlace del nitrógeno molecular (N2; N≡N) para producir dos moléculas de amonio (NH3). La FBN es llevada a cabo exclusivamente por unos pocos microorganismos pertenecientes a los dominios Bacteria y Arquea, conocidos como diazotrofos. Todos los microorganismos diazotrofos fijan N2 empleando el complejo multienzimático nitrogenasa. En función de la composición de sus cofactores metálicos, esenciales para su actividad, las nitrogenasas se clasifican en: nitrogenasas de molibdeno (Mo), vanadio (V) o de hierro (Fe). Todos los diazotrofos contienen al menos la nitrogenasa de molibdeno, mientras que otros pueden contener adicionalmente las nitrogenasas de vanadio y/o hierro. Por lo tanto, la nitrogenasa de molibdeno es la más abundante y es propuesta como el predecesor evolutivo de las nitrogenasas alternativas de vanadio y hierro. Las nitrogenasas de molibdeno, así como el conjunto de proteínas requeridas para su ensamblaje, función y regulación, están codificadas por una serie de genes conocidos como nif (“nitrogen fixation genes”). La cantidad y composición de genes nif varía en función de las necesidades fisiológicas y ecológicas de cada diazotrofo. Sin embargo, se ha establecido un mínimo de seis genes nif como criterio esencial para la fijación de nitrógeno dependiente de molibdeno (nifHDKENB). En la presente tesis se investiga la nitrogenasa de la bacteria termófila Roseiflexus sp. RS-1, que parece depender exclusivamente de los cuatro genes nifHBDK. Los componentes estructurales de la nitrogenasa NifH y NifDK, así como el componente biosintético NifB, han sido caracterizados. La demostración de la actividad in vitro de los tres componentes Nif de Roseiflexus sp. RS-1 demuestra la existencia de una ruta alternativa en la biosíntesis del cofactor de hierro y molibdeno (FeMo-co). En Roseiflexus sp. la síntesis del cofactor FeMo-co parece ser independiente de NifEN, mientras que NifDK parece tener una capacidad dual, participando en la maduración de FeMo-co así como, manteniendo su actividad nitrogenasa. Estos resultados sugieren que Roseiflexus sp. posee un complejo enzimático que se asemeja al predecesor de las nitrogenasas de molibdeno actuales antes de los eventos de duplicación y divergencia de los genes nifDK y nifEN. Además, esta tesis incluye la primera descripción de la estructura de NifB obtenida por rayos X. Debido a que la proteína NifB de Roseiflexus sp. RS-1 no pudo ser cristalizada, la estructura de su homólogo procedente de Methanotrix thermoacetophila fue finalmente resuelta en colaboración con el grupo del Dr Yvain Nicolet del “Institute de Biologie Structurale”. Se ha descrito una coordinación novedosa de uno de los cofactores [Fe4S4], constituida por dos cisteínas, una histidina y un ácido glutámico. La mutagénesis dirigida sobre residuos de NifB, así como los ensayos bioquímicos llevados a cabo en esta tesis permitieron refinar un modelo catalítico para la síntesis del precursor NifB-co, donde una región flexible delimitada por los residuos C62 y E65 tiene un papel central en la coordinación de la unión y catálisis de la molécula de SAM, y en la estabilización de los cofactores [Fe4S4]

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

Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-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. Hydrogenobacter thermophilus (Aquificae) NifH is isolated in large quantities from yeast mitochondria and fulfills NifH protein requirements for efficient N2 fixation, including electron transfer for substrate reduction, P-cluster maturation, and FeMo-co biosynthesis. H. thermophilus NifH expressed in tobacco leaves shows lower nitrogenase activity than that from yeast. However, transfer of [Fe4S4] clusters from NifU to NifH in vitro increases 10-fold the activity of the tobacco-isolated NifH, revealing that plant mitochondria [Fe-S] cluster availability constitutes a bottleneck to engineer plant nitrogenases.ISSN:2399-364

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

A Colorimetric Method to Measure in Vitro Nitrogenase Functionality for Engineering Nitrogen Fixation

Biological nitrogen fixation (BNF) is the reduction of N2 into NH3 in a group of prokaryotes by an extremely O2-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. The ARA is not well suited for analysis of large sample sets nor easily adapted to automated robotic determination of nitrogenase activities. Here, we show that a reduced sulfonated viologen derivative (S2Vred) assay can replace the ARA for simultaneous analysis of isolated nitrogenase proteins using a microplate reader. We used the S2Vred to screen a library of NifH nitrogenase components targeted to mitochondria in yeast. Two NifH proteins presented properties of great interest for engineering of nitrogen fixation in plants, namely NifM independency, to reduce the number of genes to be transferred to the eukaryotic host; and O2 resistance, to expand the half-life of NifH iron-sulfur cluster in a eukaryotic cell. This study established that NifH from Dehalococcoides ethenogenes did not require NifM for solubility, [Fe-S] cluster occupancy or functionality, and that NifH from Geobacter sulfurreducens was more resistant to O2 exposure than the other NifH proteins tested. It demonstrates that nitrogenase components with specific biochemical properties such as a wider range of O2 tolerance exist in Nature, and that their identification should be an area of focus for the engineering of nitrogen-fixing crops