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 (RjoGlgAb and RjoGlgAc) 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. RjoGlgAb and RjoGlgAc presented the "canonical" glycogen synthase activity and were actives as maltose-1P synthases, although to a different extent. Then, RjoGlgAc is a homologous enzyme to the mycobacterial GlgM, with similar kinetic behavior and glucosyl-donor preference. RjoGlgAc was two orders of magnitude more efficient to glucosylate glucose-1P than glycogen, also using glucosamine-1P as a catalytically efficient aglycon. Instead, RjoGlgAb exhibited both activities with similar kinetic efficiency and 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 relationship. Kinetic results presented herein constitute a hint to infer on polysaccharides biosynthesis in rhodococci from an enzymological point of view.

New pieces to the carbon metabolism puzzle of Nitrosomonas europaea: Kinetic characterization of glyceraldehyde-3 phosphate and succinate semialdehyde dehydrogenases.

Nitrosomonas europaea is a chemolithotroph that obtains energy through the oxidation of ammonia to hydroxylamine while assimilates atmospheric CO2 to cover the cell carbon demands for growth. This microorganism plays a relevant role in the nitrogen biogeochemical cycle on Earth but its carbon metabolism remains poorly characterized. Based on sequence homology, we identified two genes (cbbG and gabD) coding for redox enzymes in N. europaea. Cloning and expression of the genes in Escherichia coli, allowed the production of recombinant enzymes purified to determine their biochemical properties. The protein CbbG is a glyceraldehyde-3-phosphate (Ga3P) dehydrogenase (Ga3PDHase) catalyzing the reversible oxidation of Ga3P to 1,3-bis-phospho-glycerate (1,3bisPGA), using specifically NAD+/NADH as cofactor. CbbG showed ∼6-fold higher Km value for 1,3bisPGA but ∼5-fold higher kcat for the oxidation of Ga3P. The protein GabD irreversibly oxidizes Ga3P to 3Pglycerate using NAD+ or NADP+, thus resembling a non-phosphorylating Ga3PDHase. However, the enzyme showed ∼6-fold higher Km value and three orders of magnitude higher catalytic efficiency with succinate semialdehyde (SSA) and NADP+. Indeed, the GabD protein identity corresponds to an SSA dehydrogenase (SSADHase). CbbG seems to be the only Ga3PDHase present in N. europaea; which would be involved in reducing triose-P during autotrophic carbon fixation. Otherwise, in cells grown under conditions deprived of ammonia and oxygen, the enzyme could catalyze the glycolytic step of Ga3P oxidation producing NADH. As an SSADHase, GabD would physiologically act producing succinate and preferentially NADPH over NADH; thus being part of an alternative pathway of the tricarboxylic acid cycle converting α-ketoglutarate to succinate. The properties determined for these enzymes contribute to better identify metabolic steps in CO2 assimilation, glycolysis and the tricarboxylic acid cycle in N. europaea. Results are discussed in the framework of metabolic pathways that launch biosynthetic intermediates relevant in the microorganism to develop and fulfill its role in nature.