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.

Plastidic phosphoglycerate kinase from Phaeodactylum tricornutum: on the critical role of cysteine residues for the enzyme function.

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.