Characterization of Recombinant UDP- and ADP-Glucose Pyrophosphorylases and Glycogen Synthase To Elucidate Glucose-1-Phosphate Partitioning into Oligo- and Polysaccharides in Streptomyces coelicolor

Abstract

b Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo-and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP-and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V max in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V max of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate ␣-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo-and polysaccharides, as well as with antibiotic synthesis in the bacterium. Gram-positive bacteria of the genus Streptomyces exhibit a particularly complex morphological differentiation, with the formation of a highly branched substrate and aerial mycelium that generates spores from septation of the aerial hyphae (23, 33). Also characteristic of Streptomyces spp. is the occurrence of a flush secondary metabolism, with these bacteria producing almost twothirds of all known natural antibiotics (9). Streptomyces coelicolor A3(2) is the producer of three pigmented antibiotics, and it has a well-characterized genetics; these characteristics make it one of the preferred strains for research within this genus of bacteria. Although the complete genome of this S. coelicolor strain was elucidated about 10 years ago (5), advanced knowledge of the microorganism at the biochemical level is far from being reached. Most of the studies carried out with Streptomyces spp. have been biased toward the understanding of its morphological and physiological differentiation, while very little has been done on the biochemistry of the enzymes involved in the metabolic pathways.Monosaccharide interconversion and synthesis of oligo-and polysaccharides are key proces...

The Production and Utilization of GDP-glucose in the Biosynthesis of Trehalose 6-Phosphate by Streptomyces venezuelae

“…With the absence of a known sequence being associated with such an enzyme, we tested three candidate enzymes based on homology with the potentially similar GDP-mannose pyrophosphorylases. SVEN_3027 generated UDP-glucose as expected, given that it was also a homologue of GalU/GtaB from other organisms (33, 38). SVEN_3972, on the other hand, was somewhat unusual because its main activity appeared to be associated with the production of IDP-mannose (Table 3).…”

“…The enzyme was present primarily in an active dimeric form, but inactive octameric and higher oligomeric forms were also detected using size exclusion chromatography. Enzyme activity was not affected by any of the potential allosteric effectors described above, in contrast to enzymes such as ADP-glucose pyrophosphorylase from other actinomycetes (27, 33). …”

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

“…Eukaryotic UDP-Glc PPases are bigger than those found in bacteria [5,6], and the enzyme from Entamoeba histolytica (and probably from all protozoa) was recently characterized as being regulated by redox modification of critical cysteinyl residues [7]. Many bacterial UDP-Glc PPases have been characterized [8–13], and the crystallographic structures of the enzyme from Escherichia coli [14], Sphingomonas elodea [15] and Corynebacterium glutamicum [16] have been elucidated. The prokaryotic UDP-Glc PPase is a dimeric/tetrameric protein formed by a single subunit of ~35 kDa with a relatively high specific activity and specificity for Glc-1P and UTP [8–13].…”

“…Many bacterial UDP-Glc PPases have been characterized [8–13], and the crystallographic structures of the enzyme from Escherichia coli [14], Sphingomonas elodea [15] and Corynebacterium glutamicum [16] have been elucidated. The prokaryotic UDP-Glc PPase is a dimeric/tetrameric protein formed by a single subunit of ~35 kDa with a relatively high specific activity and specificity for Glc-1P and UTP [8–13]. …”

The ancestral activation promiscuity of ADP-glucose pyrophosphorylases from oxygenic photosynthetic organisms

“…Recently, we have found that the enzyme from Streptomyces coelicor has a very different specificity for activators and may fall into a new class. It is activated mainly by glucose-6-phosphate (Glc6P) and can also be activated by mannose-6-phosphate, phosphoenolpyruvate, and Fru6P [12]. The enzyme from Nitrosomonas europaea can be activated mainly by pyruvate, but also by oxaloacetate and phosphoenolpyruvate [13].…”