Abstract:

Natural and modified nucleoside-5′-monophosphates and their precursors are valuable compounds widely used in biochemical studies. Bacterial nonspecific acid phosphatases (NSAPs) are a group of enzymes involved in the hydrolysis of phosphoester bonds, and some of them exhibit phosphotransferase activity. NSAP containing Enterobacter aerogenes and Raoultella planticola whole cells were evaluated in the phosphorylation of a wide range of nucleosides and nucleoside precursors using pyrophosphate as phosphate donor. To increase the productivity of the process, we developed two genetically modified strains of Escherichia coli which overexpressed NSAPs of E. aerogenes and R. planticola. These new recombinant microorganisms (E. coli BL21 pET22b-phoEa and E. coli BL21 pET22b-phoRp) showed higher activity than the corresponding wild-type strains. Reductions in the reaction times from 21 h to 60 min, from 4 h to 15 min, and from 24 h to 40 min in cases of dihydroxyacetone, inosine, and fludarabine, respectively, were obtained.

Author affiliation: Médici, Rosario. Delft University of Technology; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Area Química. Laboratorio de Biotransformaciones; Argentina

Author affiliation: Garaycoechea, Juan I.. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Area Química. Laboratorio de Biotransformaciones; Argentina

Author affiliation: Valino, Ana Laura. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Area Química. Laboratorio de Biotransformaciones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina

Author affiliation: Lewkowicz, Elizabeth Sandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Area Química. Laboratorio de Biotransformaciones; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Iribarren, Adolfo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación en Ingeniería Genética y Biología Molecular "Dr. Hector N. Torres". Grupo Vinculado al INGEBI- Laboratorio de Biocatálisis y Biotransformaciones - LBB - UNQUI; Argentina

Abstract:

Nonspecific acid phosphatases are a group of enzymes whose activity increases the availability of exogenous and endogenous orthophosphate either through extra- or intracellular hydrolysis of phosphate compounds. Our study demonstrates the activity of acid phosphatases in the filamentous freshwater alga Stigeoclonium tenue. These enzymes were detected following a cerium-based method in which cerium was used as an orthophosphate-capture reagent. In thalli from S. tenue from the natural environment, acid phosphatases were found in the longitudinal cell wall, plasmalemma, and vacuole. In thalli from Bold’s Basal Medium culture, these enzymes were found mainly in the plasmalemma; they were scarce in the cell wall. In the thalli grown in phosphate-enriched culture medium, enzymes were found only in the plasmalemma. The low availability of orthophosphate in the medium seems to induce the transport of these enzymes to the cell wall. Its abundance, on the contrary, seems to attenuate this response without affecting the localization of acid phosphatases in the plasmalemma.

Author affiliation: Michetti, Karina Mariel. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Ficología y Micología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Author affiliation: Leonardi, Patricia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

Author affiliation: Caceres, Eduardo Jorge. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Laboratorio de Ficología y Micología; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina

Abstract:

BACKGROUND: Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. RESULTS: We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. CONCLUSIONS: The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels production

Author affiliation: Comba, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

Author affiliation: Menendez Bravo, Simón Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

Author affiliation: Arabolaza, Ana Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

Author affiliation: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentina

Abstract:

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas' disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

Author affiliation: Leyria, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Author affiliation: Fruttero, Leonardo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Author affiliation: Nazar, Magalí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Author affiliation: Canavoso, Lilian Etelvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina

Abstract:

Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. Lipin family is a Mg-dependent type I PA phosphatase,involved in de novo synthesis of neutral lipids and of phospholipids. The regulation of Lipin activity may govern the pathways by which these lipids aresynthesized and control the cellular levels ofimportant signaling lipids. On the other hand, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation: by interacting with key synthetizing enzymes it is able toincrease overall phopho- and glyco- lipid synthesis.We studied the Lipin 1β enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that Lipin 1β kcat increases around 40% in the presence of c- Fos, with no change in the Lipin 1β affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. While the c-Fos domain involved in Lipin activation is its basic domain (BD), the interaction domain is mapped to the c-Fos N-terminal. In conclusion, we provide evidence for a novel positive regulator of Lipin 1β PA phosphatase activity that is not achieved via altering its subcellular localization or affinity for membranesbut rather through directly increasing its catalytic efficiency.

Author affiliation: Cardozo Gizzi, Andres Mauricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentina

Author affiliation: Prucca, Cesar German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentina

Author affiliation: Gaveglio, Virginia Lucía. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina

Author affiliation: Renner, Marianne L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (p); Argentina

Author affiliation: Pasquaré, Susana J.. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina

Author affiliation: Caputto, Beatriz Leonor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Química Biológica de Córdoba (p); Argentina