Biochemical characterization of Neurospora crassa glycogenin (GNN), the self-glucosylating initiator of glycogen synthesis

Glycogenin acts in the initiation step of glycogen biosynthesis by catalyzing a self-glucosylation reaction. In a previous work [de Paula et al., Arch. Biochem. Biophys. 435 (2005) 112-124], we described the isolation of the cDNA gnn, which encodes the protein glycogenin (GNN) in Neurospora crassa. This work presents a set of biochemical and functional studies confirming the GNN role in glycogen biosynthesis. Kinetic experiments showed a very low GNN K(m) (4.41 microM) for the substrate UDP-glucose. Recombinant GNN was produced in Escherichia coli and analysis by mass spectroscopy identified a peptide containing an oligosaccharide chain attached to Tyr196 residue. Site-directed mutagenesis and functional complementation of a Saccharomyces cerevisiae mutant strain confirmed the participation of this residue in the GNN self-glucosylation and indicated the Tyr198 residue as an additional, although less active, glucosylation site. The physical interaction between GNN and glycogen synthase (GSN) was analyzed by the two-hybrid assay. While the entire GSN was required for full interaction, the C-terminus in GNN was more important. Furthermore, mutation in the GNN glucosylation sites did not impair the interaction with GSN.     

Long-term effects of several biochemical and physical factors on cellulose biosynthesis in callus and suspension cultures of normal and mutant barley (Hordeum vulgare L.) strains producing less cellulose

To clarify a low level of cellulose biosynthesis of thein vitro cultured cells, the effects of several biochemical factors such as carbon sources (sucrose, maltose, and UDPG), antioxidants (ascorbic acid and glutathione) and physical factors such as artificial pressure, high gravity, on the cellulose production in barley callus and suspension cultures were investigated. In the suspension culture of two barley strains, the supplement of different concentrations (0, 1.5, 3.0, and 4.5%) of sucrose or maltose into the medium for 30 days did not promote the cellulose production and 4.5% of sugar supplement was rather inhibitory in one strain. However, in the presence of sucrose at 3%, UDPG (3 or 10 mM) supplement, as a precursor for cellulose, promoted 1.2–13 fold of the production in two strains. A low concentration (3 mM) of ascorbic acid and glutathione promoted 1.5 and 1.2 fold of the production in two strains, respectively. These results suggest that low cellulose biosynthesis of thein vitro cultured cells is due to a decreased level of the UDPG in the cytosol, and that the oxidative condition of external medium impedes cellulose synthesis in some manners. Artificial pressure applied to the callus promoted 1.4 fold of the cellulose production. High gravity (5,000 or 10,000g) applied to the suspension-cultured cells by centrifugation did not cause a substantial change.     

Enzymatic activities of starch synthesis in potato tubers of different sizes

The objective of this study was to determine the relationship between tuber weight and enzymatic activities involved in tuber starch synthesis. As tuber weight increased, the activities of sucrose synthetase, UDPG pyrophosphorylase, and granular starch synthetase escalated, whereas the activities of soluble starch synthetase and ADPG pyrophosphorylase stayed constant and that of phosphorylase declined. This suggests that when samples are taken to determine specific enzymatic activities, the sampling procedure should ensure that results do not vary because of differences in the tuber weight or size distribution.     

Gene-dependent flavonoid glucosyltransferase in maize

A direct relationship between a specific gene and a specific enzyme involved in flavonoid biosynthesis is reported for the gene Bz and uridine diphosphoglucose: quercetin glucosyltransferase in maize pollen, seedlings, and seeds. Ratios are presented for specific activities of the glucosyltransferase from pollen, seed, and seedling tissues homozygous and heterozygous for Bz and homozygous for bz.Cooperative Investigations, Agricultural Research Service, United States Department of Agriculture and Missouri Agricultural Experiment Station, Columbia, Missouri 65201. Journal Series No. 7340.     

Arginine mediated purification of trehalose-6-phosphate synthase (TPS) from Candida utilis: Its characterization and regulation

Background

Trehalose is the most important multifunctional, non-reducing disaccharide found in nature. It is synthesized in yeast by an enzyme complex: trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP).

Methods

In the present study TPS is purified using a new methodology from Candida utilis cells by inclusion of 100 mM l-arginine during cell lysis and in the mobile phase of high performance gel filtration liquid chromatography (HPGFLC).

Results

An electrophoretically homogenous TPS that was purified was a 60 kDa protein with 22.1 fold purification having a specific activity of 2.03 U/mg. Alignment of the N-terminal sequence with TPS from Saccharomyces cerevisiae confirmed the 60 kDa protein to be TPS. Optimum activity of TPS was observed at a protein concentration of 1 μg, at a temperature of 37 °C and pH 8.5. Aggregation mediated enzyme regulation was indicated. Metal cofactors, especially MnCl₂, MgCl₂ and ZnSO₄, acted as stimulators. Metal chelators like CDTA and EGTA stimulated enzyme activity. Among the four glucosyl donors, the highest V_max and lowest K_m values were calculated as 2.96 U/mg and 1.36 mM when adenosine di phosphate synthase (ADPG) was used as substrate. Among the glucosyl acceptors, glucose-6-phosphate (G-6-P) showed maximum activity followed by fructose-6-phosphate (F-6-P). Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity with different glucosyl donors.

General significance

Substrate specificity, V_max and K_m values provided an insight into an altered trehalose metabolic pathway in the C. utilis strain where ADPG is the preferred substrate rather than the usual substrate uridine diphosphaphate glucose (UDPG). The present work employs a new purification strategy as well as highlights an altered pathway in C. utilis.     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

The role of UDP-glucose:hydroxycinnamate glucosyltransferases in phenylpropanoid metabolism and the response to UV-B radiation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Arabidopsis harbors four UDP-glycosyltransferases that convert hydroxycinnamates (HCAs) to 1-O-β-glucose esters, UGT84A1 (encoded by At4g15480), UGT84A2 (At3g21560), UGT84A3 (At4g15490), and UGT84A4 (At4g15500). To elucidate the role of the individual UGT84A enzymes in planta we analyzed gene expression, UGT activities and accumulation of phenylpropanoids in Arabidopsis wild type plants, ugt mutants and overexpressing lines. Individual ugt84A null alleles did not significantly reduce the gross metabolic flux to the accumulating compounds sinapoylcholine (sinapine) in seeds and sinapoylmalate in leaves. For the ugt84A2 mutant, LC/MS analysis revealed minor qualitative and quantitative changes of several HCA choline esters and of disinapoylspermidine in seeds. Overexpression of individual UGT84A genes caused increased enzyme activities but failed to produce significant changes in the pattern of accumulating HCA esters. For UGT84A3, our data tentatively suggest an impact on cell wall-associated 4-coumarate. Exposure of plants to enhanced UV-B radiation induced the UGT84A-encoding genes and led to a transient increase in sinapoylglucose and sinapoylmalate concentrations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

Sucrose synthetase from triploid quaking aspen callus

Sucrose synthetase and UDPG pyrophosphorylase were found in crude extracts of dark-grown triploid quaking aspen callus. The sucrose synthetase was like that from other plants, but it appeared to be membrane-bound. The activity also occurred in extracts prepared from callus of other tree species.     

Purification and properties of sterol: UDPG glucosyltransferase in cell culture of Digitalis purpurea

Sterol: UDPG glucosyltransferase was isolated for the first time from cell culture. Digitalis purpurea cultured cells had 2–5 times higher activity than that of the original plant. The enzyme in the particulate fraction was purified 70.2-fold from cell culture and 76-fold from the plant by cellular fractionation and column chromatography. The properties of purified enzyme from cultured cells were similar to those of the enzyme from the intact plant. The substrate specificity was the highest for a phytosterol.