Comparative study of both glycogen synthases from Bradyrhizobium japonicum

IGLESIAS AA; FERRETTI MV; ASENCION DIEZ MD; FIGUEROA CM

Workshop; Primer Workshop virtual de la Red Argentina de Tecnología Enzimática; 2021

Thebiosynthesis of α-1,4-polyglycans is a strategy widely used by living organismsfor the intracellular storage of carbon and energy. In bacteria glycogen synthesisinvolves three reactions, where ADP-glucose (ADP-Glc) catalyzed by theallosteric ADP-Glc pyrophosphorylase (EC 2.7.7.27), is used to elongate anα-1,4-glucan chain by glycogen-synthase (EC 2.4.1.21; GlgSase). Later, thepolyglucan branching enzyme (EC 2.4.1.18) incorporates the α-1,6 branches toachieve the final glucan structure.  Theelucidation of different prokaryotic genomes has shown that a significantnumber of bacteria possess more than one glgA gene, encoding GlgSase-typeglycosyltransferases. Particularly, Bradyrhizobium japonicum, aGram-negative, bar-shaped, nitrogen-fixing bacterium, has two coding sequences(bll2778 and blr6459I) for putative GlgSases in its genome.Proteins were arbitrary named GlgSase471 (encoded by bll2778) andGlgSase482 (blr6459), based on their total amino acidic residues. Thebiochemical characterization of the B. japonicum GlgSases constitutesthe main goal of this work. Both GlgSases shown an identity of 41.7% and asimilarity of 59.5% in their amino acid sequences. We built a phylogenetic treefrom a sequence alignment, comparing the two GlgSases of B. japonicumwith those of others from different organisms. The GlgSase471 and GlgSase482located in the same clade as the enzymes from enterobacteria, cyanobacteria andFirmicutes (all belonging to the family of GT5 glycosyltransferases). Instead,the enzymes from phylum Actinobacteria (GT4 glycosyltransferases withanother activity synthesizing maltose-1P) formed a separate group. Thus, thearising of questions regarding the physiological relevance of duplicatedGlgSases. So far, this topic was scarcely approached concerning bacterialglycogen metabolism. Bothbll2778 and blr6459 genes were de novo synthesized and clonedinto the vector pET28b for their recombinant expression. Both proteins wereproduced with a N-term His-tag that allowed the obtention of electrophoreticallypure GlgSase471 and GlgSase482 aliquots after purification by immobilized metalaffinity chromatography. When analyzed by analytical gel filtration procedures,both GlgSases from B. japonicum eluted as monomeric proteins. Bothenzymes were able to elongate a preformed glycogen molecule and lackedmaltose-1P synthesis activity. Results presented here constitute the first reporton the concomitant study of two prokaryotic GlgSases elongating α-glucans. Thekinetic characterization showed that GlgSase482 is two orders of magnitude moreactive (53.1 ± 0.6 U/mg) than GlgSase471 (0.35 ± 0.01 U/mg) and both arespecific for ADP-Glc, with similar apparent affinities values (KM~ 0.16 ± 0.01 mM). Regarding glycogen as a substrate, the KMvalue for GlgSase482 is 0.06 ± 0.01 mg/ml while for GlgSase471 is 0.032 ± 0.009mg/ml. The addition of 10 mM Mg2+ was essential to reach their maximalactivity, although in the absence of Mg2+ the enzymes were already actives. Theaddition of 10 mM Mn2+to the reaction mixture inhibited the activityby more than 50%.  Theanalysis of the genomic context shows that the gene encoding GlgSase482 locatedadjacent to the one coding for ADP-Glc pyrophosphorylase, then probably beingpart of a glg operon for glycogen synthesis. On the other hand, gene forGlgSase471 locates elsewhere, together with a priori unrelated genes.This agrees with the fact that GlgSase482 is the enzyme with the highestefficiency for glucan elongation. Still, further studies need to be conductedto clearly comprehend the duplication of GlgSases (but not other enzymes fromglycogen biosynthetic pathway) in B. japonicum. This will allow furtherevolutionary understanding of carbohydrate-active enzymes that proportionate tothe glucan its ultimate structure.