Abstract
AbstractThe bacterial genusRhodococcuscomprises organisms that perform an oleaginous behavior under certain growth conditions and the ratio of carbon and nitrogen availability. Thus,Rhodococcusspp have outstanding biotechnological features as microbial producers of biofuel precursors, which would be used instead of lipids from crops. It was postulated that lipid and glycogen metabolism inRhodococciare closely related. Thus, a better understanding of rhodococcal carbon partitioning requires identifying the catalytic steps redirecting sugar moieties to temporal storage molecules, such as glycogen and trehalose. In this work, we analyzed two glycosyl-transferases GT4 fromR. jostii,RjoGlgAb andRjoGlgAc, which were annotated as α-glucan-α-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were recombinantly produced inE. coliBL21 (DE3) cells, purified to near homogeneity, and kinetically characterized.RjoGlgAb andRjoGlgAc presented the “canonical” glycogen synthase (EC 2.4.1.21) activity. Besides, both enzymes were actives as maltose-1P synthases (GlgM, EC 2.4.1.342), although to a different extent. In this scenario,RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar behavior regarding kinetic parameters and glucosyl-donor (ADP-glucose) preference.RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen. Also, this rhodococcal enzyme used glucosamine-1P as a catalytically efficient aglycon. On the other hand, both activities exhibited byRjoGlgAb depicted similar kinetic efficiency and a preference for short-branched α-1,4-glucans. Curiously,RjoGlgAb presented a super-oligomeric conformation (higher than 15 subunits), representing a novel enzyme with a unique structure to function relationships. Results presented herein constitute a milestone regarding polysaccharide biosynthesis in Actinobacteria, leading to (re)discovery of methyl-glucose lipo-polysaccharide metabolism inRhodococci.
Publisher
Cold Spring Harbor Laboratory