Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs

Respuesta de preosteoblastos a compuestos de estroncio o calcio: proliferación, diferenciación, mineralización y respuesta génica global

Fundamento: Los mecanismos que desencadenan la osteogénesis todavía no están aclarados. El objetivo de este estudio fue valorar el papel de estroncio y calcio, aportados en distinto soporte molecular, como inductores de distintos mecanismos de estímulo osteoblástico, incluyendo proliferación, diferenciación y mineralización de células preosteoblásticas. Se investigó también la respuesta global genómica con la técnica de microarray. Métodos: Se diseñó un estudio experimental con células pre-osteoblásticas murinas MC3T3-E1, que fueron estimuladas durante 3 horas y 7 días. Se realizaron estudios bioquímicos y de expresión génica del genoma de ratón (Affymetrix). Resultados: El estroncio unido a ranelato (SrRn) fue el más potente inductor de la capacidad de mineralización, en comparación con los otros compuestos utilizados (2,55 veces respecto al control). Los estudios de expresión génica global mostraron que a las 3 horas cambian 2.030 genes de los cuales 1.644 genes son específicos de esta fase. Por el contrario, a 7 días de tratamiento sólo cambian 329 genes siendo específicos 147 genes. Los procesos biológicos más enriquecidos a las 3 horas fueron los involucrados en la regulación transcripcional (147 genes), procesos metabólicos (140 genes) y la fosforilación de proteínas (44 genes) entre otros, mientras que a 7 días hubo cambios relacionados con el ciclo celular (18 genes) y con el metabolismo de carbohidratos en general (12 genes). Conclusión: El estroncio unido al anión ranelato se comportó como el más potente inductor de la osteogénesis comparado con otros aniones como cloruro o hidróxidos. El estímulo a 3 horas presentó mayores cambios de expresión de genes en comparación a 7 días. Los procesos biológicos afectados pueden ser útiles para especular sobre cascadas de señalización involucradas en la activación osteoblástica y sobre nuevas dianas moleculares con fines terapéuticos

Metabolic dependency of chorismate in Plasmodium falciparum suggests an alternative source for the ubiquinone biosynthesis precursor

The shikimate pathway, a metabolic pathway absent in humans, is responsible for the production of chorismate, a branch point metabolite. In the malaria parasite, chorismate is postulated to be a direct precursor in the synthesis of p-aminobenzoic acid (folate biosynthesis), p-hydroxybenzoic acid (ubiquinone biosynthesis), menaquinone, and aromatic amino acids. While the potential value of the shikimate pathway as a drug target is debatable, the metabolic dependency of chorismate in P. falciparum remains unclear. Current evidence suggests that the main role of chorismate is folate biosynthesis despite ubiquinone biosynthesis being active and essential in the malaria parasite. Our goal in the present work was to expand our knowledge of the ubiquinone head group biosynthesis and its potential metabolic dependency on chorismate in P. falciparum. We systematically assessed the development of both asexual and sexual stages of P. falciparum in a defined medium in the absence of an exogenous supply of chorismate end-products and present biochemical evidence suggesting that the benzoquinone ring of ubiquinones in this parasite may be synthesized through a yet unidentified route

The complete genome and proteome of Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats