Characterization of a Euglena gracilis glycosyl phosphorylase from a novel carbohydrate active enzyme family

CALLONI, RODRIGO D.; MUCHUT, ROBERTINO J.; GARAY, ALBERTO S.; ARIAS, DIEGO G.; IGLESIAS, ALBERTO A.; GUERRERO, SERGIO A.

Rosario

Workshop; V Encuentro y II Workshop de la Red Argentina de Tecnología Enzimática; 2023

Red Argentina de Tecnología Enzimática

Euglena gracilis is a single-celled flagellate protist that lives in aquatic environments. E. gracilis is a microorganism capable of growing both photosynthetically, heterotrophically, and mixotrophically. Whatever the growing condition, E. gracilis manages to accumulate different bioactive compounds inside the cell, especially paramylon and wax esters, both of which are relevant due to their potential use in generating biodegradable plastics or bioethanol. Each growth condition implies the activation of specific metabolic pathways and producing different forms of carbon accumulation. Thus, studying the biochemical, functional, and regulatory properties of the enzymes involved in the synthesis and degradation of Euglenoids reserves is important. Paramylon is the main reserve polymer of E. gracilis. It is a water-insoluble β-1,3-glucan with a high degree of polymerization. There is little information about the enzymes involved in the carbon metabolism in E. gracilis. Recently, the presence of a protein in E. gracilis belonging to the family 149 of glycosyl hydrolases (EgGH149) was reported. GH149 is a new family of "carbohydrate active enzyme" (CAZyme) and is thought to group glycosyl phosphorylase. To obtain information about the function of this enzyme, we produced it recombinantly in a soluble and active form.The kinetic parameters of the enzyme in both directions (and for several substrates) were determined. EgGH149 catalyzed the condensation of glucose with glucose-1-phosphate without reaction with other sugar-1-phosphate. We also show that it can use laminaribiose (disaccharide of glucose with β-1,3 bond) and 2-deoxy-glucose with lower affinity than glucose. We do not detect enzymatic activity when evaluating other acceptors.On the other hand, we have studied the partition of Laminaribiose with inorganic phosphate and we found that the enzyme had its maximum activity at pH 7.5 and at 40 ° C. We observed that the enzyme had no activity when testing other types of disaccharides. EgGH149 efficiency decreases with increasing degree of polymerization (PD) when testing different laminarisaccharides (PD between 2 and 6) without activity towards laminarin (PD ~ 30) nor Paramylon (PD ~ 3000). Moreover, we decided to study the ability of enzyme to bind paramylon and laminarin. We did not observe binding of the EgGH149 to the substrates tested.We performed site-directed mutagenesis and kinetically characterized all the variants obtained. In this way, we identify the active site and relevant amino acids for enzymatic activity. To know the quaternary structure of the enzyme we perform a gel filtration chromatography, showing that the EgGH149 forms homodimers. With this information and the crystallized structure of another protein belonging to GH149 we made a 3D model of the protein in which we detected an oligosaccharide binding surface away from the active site.We compared the affinity of different laminarisaccharides by molecular dynamics to the active site and to the binding surface. The active site presented a decrease in affinity as the size of the laminarisaccharide increases. While the surface shows greater affinity for the hexasaccharide.Through western blot assays we show the presence of the protein in cells grown under autotrophic and heterotrophic conditions. In order to obtain information about its intracellular location, we performed a confocal microscopy assay: we observed signal recognition in the cytosol, forming hotspots near the paramylon granules regardless of the culture condition analyzed. This work provides information about the kinetic and structural behavior of this enzyme, as well as its distribution pattern in E. gracilis cells.