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The 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-1,4-glucosyl transferases, putatively involved in glycogen synthesis. Both enzymes were recombinantly produced inEscherichia 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)discover the methyl-glucose lipo-polysaccharide metabolism inRhodococciand to explore a link with lipid metabolism.

Until recently, all members of the cyanobacterial phylum were considered capable of performing oxygenic photosynthesis. This view has been questioned after the discovery of a group of presumed non-photosynthetic cyanobacteria named Melainabacteria. Using metagenomic data, we identified sequences encoding putative ADP-glucose pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase) from free-living and intestinal Melainabacteria. These genes were de novo synthesized and overexpressed in Escherichia coli. The purified recombinant proteins from the free-living and the intestinal Melainabacteria showed ADP-GlcPPase activity, with Vmax values of 2.3 and 7.1 U/mg, respectively. Both enzymes had similar affinities towards ATP (S0.5 ~0.3 mM) although the one from the intestinal source displayed a 6-fold higher affinity for glucose-1P. Both recombinant ADP-GlcPPases were sensitive to allosteric activation by glucose-6P (A0.5 ~0.3 mM), and to inhibition by Pi and ADP (I0.5 between 0.2 to 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. To the best of our knowledge, this is the first biochemical characterization of an active enzyme from Melainabacteria, offering further data to discussions regarding their phylogenetic position. This work contributes to a better understanding regarding the evolution of allosteric mechanisms in ADP-GlcPPases, an essential enzyme for the synthesis of glycogen in prokaryotes and starch in plants.