A Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites.

Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of ß-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.

Characterization of three beta-galactoside phosphorylases from Clostridium phytofermentans: discovery of d-galactosyl-beta1->4-l-rhamnose phosphorylase.

We characterized three d-galactosyl-beta1-->3-N-acetyl-d-hexosamine phosphorylase (EC 2.4.1.211) homologs from Clostridium phytofermentans (Cphy0577, Cphy1920, and Cphy3030 proteins). Cphy0577 and Cphy3030 proteins exhibited similar activity on galacto-N-biose (GNB; d-Gal-beta1-->3-d-GalNAc) and lacto-N-biose I (LNB; d-Gal-beta1-->3-d-GlcNAc), thus indicating that they are d-galactosyl-beta1-->3-N-acetyl-d-hexosamine phosphorylases, subclassified as GNB/LNB phosphorylase. In contrast, Cphy1920 protein phosphorolyzed neither GNB nor LNB. It showed the highest activity with l-rhamnose as the acceptor in the reverse reaction using alpha-d-galactose 1-phosphate as the donor. The reaction product was d-galactosyl-beta1-->4-l-rhamnose. The enzyme also showed activity on l-mannose, l-lyxose, d-glucose, 2-deoxy-d-glucose, and d-galactose in this order. When d-glucose derivatives were used as acceptors, reaction products were beta-1,3-galactosides. Kinetic parameters of phosphorolytic activity on d-galactosyl-beta1-->4-l-rhamnose were k(cat) = 45 s(-1) and K(m) = 7.9 mm, thus indicating that these values are common among other phosphorylases. We propose d-galactosyl-beta1-->4-l-rhamnose phosphorylase as the name for Cphy1920 protein.

Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

Genetic deficiencies of the glycogen phosphorylase system.

Several types of glycogen storage disease attributable to a deficiency of phosphorylase or phosphorylase kinase have been described. These diseases have been divided according to clinical symptoms, mode of inheritance, and affected tissue. However, this classification is questionable, as the clinical symptoms of these different diseases are similar, the mode of inheritance is often difficult to establish, and the biochemical assays are subject to several technical problems. A better classification would be based upon the identification of mutations in the respective disease genes. The molecular heterogeneity, however, is large, and at least 10 genes are involved. Mutations have been found in the muscle phosphorylase gene in patients with muscle phosphorylase deficiency, in the gene encoding the liver alpha subunit of phosphorylase kinase in patients with X-linked liver glycogenosis, and in the gene for the muscle alpha subunit of phosphorylase kinase in a patient with muscle phosphorylase kinase deficiency. We review here the different deficiencies of the phosphorylase system.

Purification of two forms of bovine liver glycogen phosphorylase b with distinct subunit composition.

The procedures for the purification of two forms of bovine liver glycogen phosphorylase b are described. Both forms showed a single band in nondenaturing gel electrophoresis. Gel electrophoresis in the presence of sodium dodecyl sulfate produced a single-band pattern for one of the enzyme forms (phosphorylase b1) and a triple-band pattern for the other (phosphorylase b3). Molecular weights associated with these bands were 97 kDa in the first case and 97, 55, and 40 kDa in the second. The yield from 1 kg of liver was approximately 10 mg for phosphorylase b1 and 140 mg for phosphorylase b3. The specific activity was 40-44 U/mg in both cases. As phosphorylase b1 is composed of just one kind of monomer, it is a novel bovine liver phosphorylase b structure.

Glycogen phosphorylase isozyme pattern in rat liver and isolated non-parenchymal liver cells.

The isozyme distribution of glycogen phosphorylase (EC 2.4.1.1) was studied in adult rat liver and isolated non-parenchymal liver cells by means of immuno-titration. In adult rat liver, the L-type of glycogen phosphorylase was found to dominate, whereas the heterogeneous non-parenchymal liver cell population apparently contains all three glycogen phosphorylase isozymes.

Identification of two forms of glycogen phosphorylase in Dictyostelium.

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.

Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues.

Glycogen phosphorylase has at least three isoenzymes, i.e. muscle-, liver-, and brain-types. Antibodies have been raised against highly purified isoenzymes from rat muscle, liver and brain and found to react specifically to extracts from human muscle, liver and brain, respectively. Using these antibodies and the unlabelled antibody-enzyme method, each of the three isoenzymes has been localized in both rat and human tissues.