Kinetic and structural characterization of a glycosyl phosphorylase from Euglena gracilis

CALLONI, RD; MUCHUT, RJ; ARIAS, DG; IGLESIAS, AA; GUERRERO, SA

Salta

Congreso; SAIB LV Reunión Anual; 2019

SAIB

Euglena gracilis is a fresh water protist with a large metabolic capacity because it is able to grow photosynthetically or heterotrophically. E. gracilis is a microorganism of interest in biotechnology and biomedicine due to its ability to generate bioproducts such as polysaccharides, polyunsaturated fatty acids, vitamins, wax esters and other metabolites. 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 how E. gracilis is able to metabolize this polymer. 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. Glycosyl phosphorylases can catalyze the degradation / synthesis of β-1,3-glucan. The kinetic parameters of the enzyme in both senses (and for several substrates) were determined. We have studied the partition of a disaccharide of glucose with β-1,3 bond (Laminaribiose or Lam2) with inorganic phosphate and we found that the enzyme had its maximum activity at pH 7.5 and at 40 ° C (kcat of 9.1 s-1 and a Km values of 1.57 mM for inorganic phosphate and 1.24 mM for the Lam2). We observed that the enzyme had no activity when testing other types of disaccharides. The influence of polymerization degree (PD) was evaluated, being the efficiency of the enzyme 10-fold lower for both laminaritetrose (PD=4) and laminarihexose (PD=6). No activity using laminarin (PD=30) as substrate was detected. On the other hand, EgGH149 catalyzed the condensation of glucose with glucose-1-Phosphate (kcat 1.32 s-1 and Km 1.81 for the glucose). We also carry out promiscuity tests for sugar phosphate and free sugar, finding that it is specific for the use of glucose-1-Phosphate without reaction with other sugar-1-phosphate. We also show that it is capable of using Lam2 and 2-deoxy-glucose with lower affinity than glucose. We do not detect enzymatic activity when evaluating other acceptors. In order to know the quaternary structure of the enzyme we perform a gel filtration chromatography, showing that the EgGH149 forms homodimers in agreement with previous reports for the same enzyme from other sources. 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 a laminarihexose binding surface away from the active site. In this regard, 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 recently obtained rabbit serum antibodies immunized against the protein of interest that will help to determine its cellular location. This result contributes to the right assignation structure - function for this polypeptide, being necessary further works for in depth analyzes the functionality of EgGH149 in E. gracilis.