Enzymatic production of novel α-1,4-glucans with glucosamine moieties

IGLESIAS MJ; IGLESIAS AA; ASENCION DIEZ MD

Rosario, Argentina

Workshop; V Encuentro y Segundo Workshop de la Red Argentina de Tecnología Enzimática (RedTEz); 2023

Red Argentina de Tecnologia Enzimatica (RedTEz)

The use of synthetic strategies to obtain glycans in vitro by combining purified enzymes, substrates,and optimal conditions is known as cell-free glycobiology. The composition of enzymatic systemscan be directly controlled, and the limitations of biological membranes or cell viability requirementscan be avoided. A source to improve the availability of biocatalytic tools for synthetic glycobiologyis the study and characterization of enzymes related to carbohydrate metabolism and theirpromiscuity -ability to use alternative substrates-. In this work, an ensemble of bacterial enzymescapable of channeling glucosamine (GlcN) subunits to glycogen molecules was studied. Thisarrangement is composed of three enzymes: an ADP-glucose pyrophosphorylase (GlgC, EC 2.7.7.27),a GT4-type maltosyl-1P synthase (GlgM, EC 2.4.1.142), and a maltosil-transferase (GlgE, EC2.4.16.99). ADP-glucose is synthetized by GlgC from ATP and glucose-1P. GlgM uses this ADP-Glcand another molecule of Glc-1P to form maltose-1P, which is used as substrate by GlgE to elongatea pre-formed α-1,4-Glc chain into two glycosidic moieties. We previously found that the GlgM fromRhodococcus jostii (RjoGlgM) is capable of use GlcN-1P as alternative substrate, producing aputative GlcN containing hetero-disaccharide-1P (Glc-α-1,4-GlcN-1P). We then carried out a coupledenzymatic assay adding GlgE from Streptomyces coelicolor (ScoGlgE) to the GlgM reaction mixture.Thereby, the GlcN hetero-disaccharide, synthetized in situ by RjoGlgM, could be used by ScoGlgE toelongate glycogen, releasing a phosphate molecule that can be measured. Surprisingly, the ScoGlgEactivities with either the canonical (Mal-1P) or the alternative (Glc-α-1,4-GlcN-1P) substrates were inthe same order of magnitude (~1 U/mg). Additionally, we reported that GlgC from Geobacillusstearothermophilus (GstGlgC/GlgD) catalyzes GlcN-1P utilization alternatively to Glc-1P with thehighest catalytic activity reported among several studied enzymes (~10 U/mg). Thus, GstGlgC/GlgDand RjoGlgM enzymatic assemble was studied with ATP and GlcN-1P as substrates measuring ADP release. This coupled reaction with GlcN showed a 10% of the canonical activity, proving thesynthesis of a homo-amino-disaccharide-1P molecule (GlcN-α-1,4-GlcN-1P). Finally, the ensemble of the three enzymes (GstGlgC/GlgD, RjoGlgM and ScoGlgE) was studied using GlcN-1P, ATP, andglycogen as the final acceptor. Activity assays indicate that the reaction cascade presents activityand glycogen molecules are modified by the addition of GlcN disaccharides. The enzymatic systemwas also studied with GlcN and different aglycons (maltose, maltotriose, and starch). Similaractivities were reported in all cases. Overall, the study indicates that the system presents substratepromiscuity not only for GlcN-1P but also for different α-glucans. The enzymatic system reported isa novel strategy to study enzymes generating in situ their substrates. This strategy allowed to studyenzymatic promiscuity by generating molecules not described in nature so far. These findingsprovide insights into the design of new biotechnological tools to produce glucans (oligo- and polysaccharides) in vitro with added α-1,4-amino sugars.