IAL   21557
INSTITUTO DE AGROBIOTECNOLOGIA DEL LITORAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Sucrose Metabolism in Nitrosomonas europaea
Autor/es:
FERRETTI MV; FIGUEROA CM; BALLICORA MA; ASENCION DIEZ MD; IGLESIAS AA
Reunión:
Congreso; LVI SAIB Meeting - XV SAMIGE Meeting; 2020
Institución organizadora:
Sociedad Argentina de Investigaciones Bioquimicas - Sociedad Argentina de Microbiologia General
Resumen:
n plants and cyanobacteria, sucrose synthesis occurs by the sequential action of sucrose-phosphate synthase (SPS, EC 2.4.1.14), catalyzing the conversion of fructose-6P and UDP-glucose to sucrose-6P, which is later dephosphorylated by sucrose-6P phosphatase (SPP, EC 3.1.3.24) to release free sucrose. The disaccharide can also be cleaved by UDP to form UDP-glucose and fructose by sucrose synthase (SucSase, EC 2.4.1.13) in a reversible reaction. Genomic and phylogenetic analyses revealed sucrose-related genes in the nitrifying prokaryote Nitrosomonas europaea, which obtains its energy for growth from ammonia oxidation. N. europaeais a facultative chemolithoautotrophic bacterium that grows either autotrophic or heterotrophically when the carbon source is CO2or fructose, respectively. Also, N. europaeais moderately halotolerant. It was detected in samples from disposal plants, freshwater habitats, and fertilized agricultural soils, making it suitable for potential biotechnological applications, such as bioremediation. We found that N. europaeahas a sequence coding for an ~80 kDa protein highly homologous to SPS type II (possessing both SPS and SPP domains) adjacent to the gene encoding SucSase. The latter was already studied in our group,while the biochemical characterization of the N. europaea SPS type II constitutes the main goal of this work. The recombinant protein His6-SPS-SPP was obtained after heterologous expression in Escherichia coliand purification by immobilized metal affinity chromatography. In presence of 10 mM UDP-glucose and 2 mM fructose-6P, the enzyme displayed low SPS (0.065 U/mg) and SPP (0.012 U/mg) activities. Alternatively, both domains were recombinantly produced as individual SPS and SPP proteins. Curiously, the two single enzymes were highly active, thus allowing their kinetic characterization. SPS showed activity with UDP-glucose (Vmax0.33 U/mg) and ADP-glucose (Vmax0.74 U/mg), whereas the KMfor UDP-glucose was 5.2-fold lower than for ADP-glucose. The KMfor fructose-6P was similar when using ADP-glucose or UDP-glucose. The C-terminal domain (SPP) showed a Vmaxof 30 U/mg, with a KM of 0.18 mM for sucrose-6P. To further understand the N. europaea SPS type II?s physiological role, immunodetection assays against the SPP domain were conducted in crude extracts from the bacterium grown with fructose as the carbon source. Results indicate that the enzyme is present in its low active, complete SPS-SPP ~80 kDa form. Our results contribute to understand N. europaeabiochemistry further and provide new pieces for the sucrose metabolism puzzle. Still, questions need to be answered to fulfill the complete scenario of the disaccharide role in non-photosynthetic organisms.