RESUMEN
Some microbial eukaryotes, such as the extremophilic red alga Galdieria sulphuraria, live in hot, toxic metal-rich, acidic environments. To elucidate the underlying molecular mechanisms of adaptation, we sequenced the 13.7-megabase genome of G. sulphuraria. This alga shows an enormous metabolic flexibility, growing either photoautotrophically or heterotrophically on more than 50 carbon sources. Environmental adaptation seems to have been facilitated by horizontal gene transfer from various bacteria and archaea, often followed by gene family expansion. At least 5% of protein-coding genes of G. sulphuraria were probably acquired horizontally. These proteins are involved in ecologically important processes ranging from heavy-metal detoxification to glycerol uptake and metabolism. Thus, our findings show that a pan-domain gene pool has facilitated environmental adaptation in this unicellular eukaryote.
Asunto(s)
Adaptación Fisiológica/genética , Evolución Molecular , Transferencia de Gen Horizontal , Genes Arqueales , Genes Bacterianos , Genoma de Planta/genética , Rhodophyta/genética , Rhodophyta/microbiología , Adenosina Trifosfatasas/genética , Archaea/clasificación , Archaea/genética , Bacterias/clasificación , Bacterias/genética , ADN de Algas , Filogenia , Rhodophyta/fisiologíaRESUMEN
Treatises on extremophiles are frequently focused on organisms belonging to the Archaea and Eubacteria kingdoms. However, a significant number of eukaryotes, both unicellular and multicellular, have evolved to live and thrive in extreme environments. Although less is known about eukaryotic life in extreme environments in comparison to prokaryotic extremophiles, advances in genomics and in comprehensive, high-throughput metabolic profiling techniques have provided new insight into the metabolic adaptations of eukaryotes living under extreme conditions. In this review, we will provide an overview of extremophilic life forms with emphasis on eukaryotes and we will compare metabolic adaptations in different eukaryotic extremophiles to identify generalities and specializations in adaptation to life under extreme conditions. Special emphasis will be devoted to the thermoacidophilic unicellular red alga Galdieria sulphuraria (Cyanidiaceae) as one example of a eukaryotic extremophile.
Asunto(s)
Aclimatación/fisiología , Células Eucariotas/metabolismo , Redes y Vías Metabólicas , Animales , Procesos Autotróficos , Eucariontes/metabolismo , Genómica , Humanos , Plantas/metabolismoRESUMEN
Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.
Asunto(s)
Nitrato Reductasas/química , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Sitios de Unión , Tampones (Química) , Catálisis , Reductasas del Citocromo/química , Citocromos b/química , Relación Dosis-Respuesta a Droga , Electrones , Ferricianuros/química , Proteínas Fúngicas/metabolismo , Glicerol/farmacología , Cinética , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Oxidación-Reducción , Paraquat/química , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Protones , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Sacarosa/farmacología , Viscosidad , Zea mays/metabolismoRESUMEN
Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.
Asunto(s)
Células Eucariotas/enzimología , Nitrato Reductasas/química , Nitrato Reductasas/metabolismo , Nitratos/metabolismo , Pichia/enzimología , Animales , Arginina/química , Sitios de Unión/fisiología , Agua Corporal/química , Pollos , Cristalografía por Rayos X , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Molibdeno/química , NADP/química , NADP/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de Aminoácido , Triptófano/químicaRESUMEN
NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.