CONICET | Buscador de Institutos y Recursos Humanos

Natural occurring polysaccharides(starch, cellulose, chitosan) are widely distributed in the biosphere and canbe considered as important biomass resources, constituting molecules ofpotential utility as biomaterials. Currently, glucans such as starch andglycogen are substrates for modifications and substitutions that allow theiruse in different biomedical applications, or in the food, pharmaceutical andcosmetic industries. Bacteria are a sustainable source for obtaining glucans tobe modified, instead of using those sources for food, such as plants and/oranimal tissues.In our group we have studied the kinetic,regulatory and structural aspects of several enzymes of bacterial carbohydratemetabolism, particularly the classic pathway for glycogen synthesis. The latteruses ADP-glucose (ADP-Glc) as the glucosyl donor to elongate the glucan in onesingle moiety. In recent years, an alternative route where glycogen iselongated into two glycosidic subunits from the donor maltose-1P was described.The key enzyme in the pathway is the maltosyl-transferase GlgE (EC 2.4.16.99),which was predicted to be in 14% of known bacterial genomes, coexisting withthe classical pathway. The maltosyl donor can be synthesized by a the GT4-typeglycosyl-transferase, GlgM (EC 2.4.1.142), that synthesizes maltose-1P fromADP-Glc and Glc-1P. So far, GlgM and GlgE have been characterized only from mycobacteriaand the behavior of the homologous enzymes from other sources is unknown. Thus,we focused in to increase the availability of kinetic data for these enzymes,studying the GlgM and GlgE from bacteria of biotechnological interest, such as Streptomycescoelicolor (Sco) and Rhodococcus jostii (Rjo). To doso, we addressed the joint analysis of GlgE with GlgM together as a singlecatalytic block. This strategy allowed us to analyze a possible channeling ofsubstrates, since it has been reported that the intracellular accumulation ofmaltose-1P can be toxic. As well, the coupled assay for in situ maltose-1Pgeneration resulted in the development of a molecular tool for GlgE studiescircumventing the scarce availability of the substrate.A derivation of the resultsobtained from the characterization of the GlgM/GlgE pair was the identificationof a combination of biocatalysts (RjoGlgM/ScoGlgE) which wereincorporated to a cell-free synthesis methodology of novel glycans. In thatregard, we exploited the ability both GlgM to use different hexose-1Ps and thepromiscuity of the GH13 family of glycosyl transferases, to which GlgE belongs.Thus, we propose the GlgM/GlgE block may be located as a cornerstone in thedevelopment of strategies for the production of biopolymers with potential use asbiomaterials.