Study of two glycosyltransferases related to polysaccharide biosynthesis in Rhodococcus jostii RHA1.

The bacterial genus Rhodococcus comprises organisms performing oleaginous behaviors under certain growth conditions and ratios of carbon and nitrogen availability. Rhodococci are outstanding producers of biofuel precursors, where lipid and glycogen metabolisms are closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying catalytic steps redirecting sugar moieties to storage molecules. Here, we analyzed two GT4 glycosyl-transferases from Rhodococcus jostii ( Rjo GlgAb and Rjo GlgAc) annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were produced in Escherichia coli cells, purified to homogeneity, and kinetically characterized. Rjo GlgAb and Rjo GlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, Rjo GlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. Rjo GlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, Rjo GlgAb exhibited both activities with similar kinetic efficiency and preference for short-branched α-1,4-glucans. Curiously, Rjo GlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure-to-function relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.