Producción CyT
Biocell - Cell-free glycobiology: enzymatic modification of alpha-glucans with glucosamine moieties
Congreso
Fecha:
2022Editorial y Lugar de Edición:
Instituto de Histología y Embriología; Centro Argentino de Información Científica y Tecnológica; Universidad Nacional de CuyoISSN:
1667-5746Resumen *
Cell-freeglycobiology is the application of synthetic strategies to obtain glycans invitro combining purified enzymes, substrates and optimal conditions. This modernfield allows the direct control of the system composition and avoids thelimitations of the biological membranes or cell viability requirements,favoring real-time monitoring and process automatization. The study of enzymes relatedto carbohydrate metabolism and their promiscuity is a source to improve theavailability of biocatalytic tools for synthetic glycobiology. In orderto modify α-glucans, we analyzed an ensemble of bacterial enzymes capable of channellingglucosamine (GlcN) subunits to glycogen molecules. This arrangement is composedby three enzymes: an ADP-glucose pyrophosphorylase (GlgC, EC 2.7.7.27) thatforms ADP-Glc from glucose-1P (Glc-1P) and ATP; a GT4-type maltosyl-1P synthase(GlgM, EC 2.4.1.342) that uses Glc-1P and ADP-Glc to form maltose-1P; and amaltosyl transferase (GlgE, EC 2.4.16.99) elongating glycogen molecules in twoglycosidic moieties using maltose-1P. We previously found that the GlgM from Rhodococcusjostii (RjoGlgM) is capable of use GlcN-1P as alternative substrate,reporting an enzymatic activity of 70 U/mg. This reaction would produce aputative GlcN containing hetero-disaccharide-1P (Glc-α-1,4-GlcN-1P). We thencarried out a coupled enzymatic assay adding GlgE from Streptomycescoelicolor (ScoGlgE) to the reaction mixture. Thereby, the GlcNhetero-disaccharide, synthetized in situ by RjoGlgM, couldbe used by ScoGlgE to elongate glycogen, releasing a phosphate moleculethat can be measured. Surprisingly, the ScoGlgE activities with eitherthe canonical (maltose-1P) or the alternative (Glc-α-1,4-GlcN-1P) substrateswhere in the same order of magnitude (~5 U/mg). Additionally, we reported thatGlgC from Geobacillus stearothermophilus (GstGlgCD) catalyzesGlcN-1P utilization alternatively to Glc-1P with the highest catalytic activityreported among several studied enzymes (10-12U/mg). Thus, another system was studied composed by GstGlgCD and RjoGlgMenzymes using ATP and GlcN-1P. We measured the formation of ahomo-amino-disaccharide-1P molecule by quantifying ADP release, suggesting the synthesisof GlcN-α-1,4-GlcN-1P. Finally, we assayed the ensemble of the three enzymesusing as substrates GlcN-1P, ATP and glycogen as final acceptor. Ourpreliminary results indicate that glycogen molecules are modified by the additionof terminal GlcN moieties. We also studied the enzymatic system with Glc-1P anddifferent final acceptors (maltose, maltotriose and starch). In all cases,similar activities were obtained to the analyzed aglycons. In a whole view, resultsindicate that the system presents substrate promiscuity for GlcN-1P and differentα-glucans. The work provides insights into the design of new biotechnologicaltools to produce glucans (oligo- and poly-saccharides) with added α-1,4-amino sugars, being moleculesnot described in nature so far. Información suministrada por el agente en SIGEVAPalabras Clave
glucosaminestarchglycogenglucosamine-1P