Characterization of a putative metal-dependent PTP-like phosphatase from Lactobacillus helveticus 2126 / Caracterización de una supuesta fosfatasa similar a PTP dependiente de metales de Lactobacillus helveticus 2126

To date, there are very limited reports on sequence analysis and structure-based molecular modeling of phosphatases produced by probiotic bacteria. Therefore, a novel protein tyrosine-like phosphatase was characterized from L. helveticus 2126 in this study. The purified bacterial phosphatase was subjected to mass spectrometric analysis, and the identity of constructed sequence was analyzed using peptide mass fingerprint. The 3-D structure of protein was elucidated using homology modeling, while its stability was assessed using Ramachandran plot, VERIFY 3D, and PROCHECK. The bacterium produced an extracellular phosphatase of zone diameter 15 ± 0.8 mm on screening medium within 24 h of incubation. This bacterial phosphatase was highly specific towards sodium phytate as it yielded the lowest Km value of 299.50 ± 4.95 μM compared to other phosphorylated substrates. The activity was effectively stimulated in the presence of zinc, magnesium, and manganese ions thereby showing its PTP-like behavior. The phosphatase showed a molecular mass of 43 kDa, and the corresponding M/Z ratio data yielded 46% query coverage to Bacillus subtilis (3QY7). This showed a 61.1% sequence similarity to Ligilactobacillus ruminis (WP_046923835.1). The final sequence construct based on these bacteria showed a conserved motif “HCHILPGIDD” in their active site. In addition, homology modeling showed a distorted Tim barrel structure with a trinuclear metal center. The final model after energy minimization showed 90.9% of the residues in the favorable region of Ramachandran’s plot. This structural information can be used in genetic engineering for improving the overall stability and catalytic efficiency of probiotic bacterial phosphatases.(AU)

Integration of virtual screening and susceptibility test to discover active-site subpocket-specific biogenic inhibitors of Helicobacter pylori shikimate dehydrogenase

Shikimate dehydrogenase (HpSDH) (EC 1.1.1.25) is a key enzyme in the shikimate pathway of Helicobacter pylori (H. pylori), which catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. Targeting HpSDH has been recognized as an attractive therapeutic strategy against H. pylori infection. Here, the catalytic active site in the crystal structure of HpSDH in complex with its substrate NADPH and product shikimate was examined in detail; the site can be divided into three spatially separated subpockets that separately correspond to the binding regions of shikimate, NADPH dihydronicotinamide moiety, and NADPH adenine moiety. Subsequently, a cascading protocol that integrated virtual screening and antibacterial test was performed against a biogenic compound library to identify biologically active, subpocket-specific inhibitors. Consequently, five, eight, and six promising compounds for, respectively, subpockets 1, 2, and 3 were selected from the top-100 docking-ranked hits, from which 11 compounds were determined to have high or moderate antibacterial potencies against two reference H. pylori strains, with MIC range between 8 and 93 μg/mL. It is found that the HpSDH active site prefers to accommodate amphipathic and polar inhibitors that consist of an aromatic core as well as a number of oxygen-rich polar/charged substituents such as hydroxyl, carbonyl, and carboxyl groups. Subpockets 1- and 2-specific inhibitors exhibit a generally higher activity than subpocket 3-specific inhibitors. Molecular dynamics simulations revealed an intense nonbonded network of hydrogen bonds, π-π stacking, and van der Waals contacts at the tightly packed complex interfaces of active-site subpockets with their cognate inhibitors, conferring strong stability and specificity to these complex systems. Binding energetic analysis demonstrated that the identified potent inhibitors can target their cognate subpockets with an effective selectivity over noncognate ones

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The role of sirtuns in cellular homeostasis

Sirtuins are evolutionarily conserved nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacylases or ADP-ribosyltransferases. These cellular enzymes are metabolic sensors sensitive to NAD+ levels that maintain physiological homeostasis in the animal and plant cells (AU)

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La diversidad funcional de proteína fosfatasa-1 y el papel de nuevas subunidades reguladoras / The functional diversity of protein phosphatase-1 and the role of new regulatory subunits

La fosforilación reversible de proteínas es un mecanismo que regula una gran variedad de procesos celulares y en particular la transmisión de señal. La proteína fosfatasa-1 (PP1) representa el mayor grupo de serina/treonina proteína fosfatasas. PP1 regula el metabolismo del glucógeno, el transporte intracelular, la contracción muscular, la síntesis proteica y la división celular. Esta diversidad funcional es consecuencia de la unión de un pequeño número de subunidades catalíticas con una amplia diversidad de subunidades reguladoras. Describimos aquí la proteína SIPP1, un factor del procesamiento de pre-mRNA que se une a PP1 y a la proteína PQBP-1. Dado que mutaciones en PQBP-1 es una de las causas del síndrome de retraso mental ligado al cromosoma X y que por otra parte, PQBP-1 se une a ataxina- 1 y a otras proteínas con extensiones de poliglutamina causantes de enfermedades neurodegenerativas, SIPP-1 puede estar implicado en la patogénesis de estas enfermedades

Reversible protein phosphorylation is a mechanism that regulates many cellular processes and in particular signal transduction. Protein phosphatase-1 (PP1) is the major group of serin/threonin protein phosphatases. PP1 regulates glycogen metabolism, intracellular transport, muscle contraction, protein synthesis and cell division. The functional diversity of PP1 is due to a small number of catalytic subunits which bind to a large number of regulatory subunits. We describe here the identification of SIPP1, a new interactor, which is an splicing factor of premRNA ands binds also PQBP-1 protein. Since mutations in PQBP-1 have been reported in cases of X-linked mental retardation and PQBP-1 binds ataxin-1 and other poliglutamine disease proteins SIPP1 could be an element in the pathogenesis of neurodegenerative disease