DEVELOPMENT OF AN ENZYMATIC ENSEMBLE TO MODIFY α-GLUCANS WITH GLUCOSAMINE MOIETIES

IGLESIAS MJ; IGLESIAS AA; STORANI A; ASENCION DIEZ MD; GUERRERO SA

Florianopolis

Simposio; 30th International Carbohydrate Symposium (Virtual); 2022

Cell-free glycobiology is a recently developedfield, involving a synthetic biology strategy with purified enzymes to obtainglycans in vitro. Glycans can be oligo- or poly-saccharides difficult toisolate from or even absent in natural sources. An important source to obtainnew biotechnological tools for glycan synthesis relies in studying the promiscuityof carbohydrate-related enzymes. Thus, we analyzed substrate promiscuity inseveral bacterial enzymes linked to glycogen metabolism. We found a combinationof three catalyzers able to canalize glucosamine (GlcN) subunits to anα-1,4-glucan molecule. The core components in this assembly are twoactinobacterial enzymes: the GT4-type maltosyl-1P synthase (EC 2.4.1.142, GlgM)and the maltosyl transferase GlgE (EC 2.4.16.99). The former producesmaltose-1P using ADP-glucose (ADP-Glc) and Glc-1P as glucosyl donor andacceptor, respectively; the latter elongates glucan molecules in two glycosidicmoieties. We found that the GlgM from Rhodococcus jostii (RjoGlgM)depicts the ability to use glucosamine-1P (GlcN-1P) as alternative substrate(activity about 70 U/mg) and an efficiency 3-fold lower than the canonicalsubstrate Glc-1P. Then, a putative hetero-disaccharide-1P (Glc-α-1,4-GlcN-1P)could be obtained. We then analyzed a coupled enzymatic assayincorporating the GlgE from Streptomyces coelicolor (ScoGlgE) tothe reaction mixtures of RjoGlgM. We “fed” the system with RjoGlgMsubstrates (ADP-Glc and GlcN-1P or Glc-1P), and measured the Pi release afterthe disaccharide transfer by ScoGlgE, using glycogen as the aglyconmodel. Surprisingly, ScoGlgE was able to elongate a glycogen moleculewith the putative amino-disaccharide-1P produced by RjoGlgM. The ScoGlgEactivities where in the same order of magnitude (~ 5 U/mg) with either 2 mMGlc-1P or GlcN-1P. In addition, we recently reported that the ADP-Glc pyrophosphorylase(EC 2.7.7.27) from Geobacillus stearothermophilus (GstADP-GlcPPase)catalyzes GlcN-1P utilization alternatively to its canonical substrate Glc-1Pwith similar activities (10-12 U/mg). This GstADP-GlcPPase reaction withATP and GlcN-1P would produce ADP-GlcN. Thus, we assayed another coupledsystem, combining GstADP-GlcPPase and RjoGlgM enzymes usingGlcN-1P and ATP as substrates and measuring the release of ADP, afterdisaccharide-P (putatively, GlcN-α-1,4-GlcN-1P) synthesis by RjoGlmM.Indeed, the enzyme showed activity values of 2 U/mg, thus strongly suggestingthe formation of a homo-amino-disaccharide-1P after the concomitant activity ofGstADP-GlcPPase and RjoGlmM. Finally, we arranged the threeenzymes to work together, using glycogen as a final acceptor of theamino-disaccharides-P. Our preliminary results support the bound of GlcNmoieties to a glycogen molecule. The work provides insights into the design of moleculartools to produce a glucan with α-1,4-amino moieties, a molecule notoccurring in nature.