Granule-bound starch synthase I in isolated starch granules elongates malto-oligosaccharides processively.
Isoforms of starch synthase belonging to the granule-bound starch synthase I (GBSSI) class synthesize the amylose component of starch in plants. Other granule-bound isoforms of starch synthase, such as starch synthase II (SSII), are unable to synthesize amylose. The kinetic properties of GBSSI and SSII that are responsible for these functional differences have been investigated using starch granules from embryos of wild-type peas and rug5 and lam mutant peas, which contain, respectively, both GBSSI and SSII, GBSSI but not SSII and SSII but not GBSSI. We show that GBSSI in isolated granules elongates malto-oligosaccharides processively, adding more than one glucose molecule for each enzyme-glucan encounter. Granule-bound SSII can elongate malto-oligosaccharides, but has a lower affinity for these than GBSSI and does not elongate processively. As a result of these properties GBSSI synthesizes longer malto-oligosaccharides than SSII. The significance of these results with respect to the roles of GBSSI and SSII in vivo is discussed. (+info)
Cloning, expression and characterization of YSA1H, a human adenosine 5'-diphosphosugar pyrophosphatase possessing a MutT motif.
The human homologue of the Saccharomyces cerevisiae YSA1 protein, YSA1H, has been expressed as a thioredoxin fusion protein in Escherichia coli. It is an ADP-sugar pyrophosphatase with similar activities towards ADP-ribose and ADP-mannose. Its activities with ADP-glucose and diadenosine diphosphate were 56% and 20% of that with ADP-ribose respectively, whereas its activity towards other nucleoside 5'-diphosphosugars was typically 2-10%. cADP-ribose was not a substrate. The products of ADP-ribose hydrolysis were AMP and ribose 5-phosphate. K(m) and k(cat) values with ADP-ribose were 60 microM and 5.5 s(-1) respectively. The optimal activity was at alkaline pH (7.4-9.0) with 2.5-5 mM Mg(2+) or 100-250 microM Mn(2+) ions; fluoride was inhibitory, with an IC(50) of 20 microM. The YSA1H gene, which maps to 10p13-p14, is widely expressed in all human tissues examined, giving a 1.4 kb transcript. The 41.6 kDa fusion protein behaved as an 85 kDa dimer on gel filtration. After cleavage with enterokinase, the 24.4 kDa native protein fragment ran on SDS/PAGE with an apparent molecular mass of 33 kDa. Immunoblot analysis with a polyclonal antibody raised against the recombinant YSA1H revealed the presence of a protein of apparent molecular mass 33 kDa in various human cells, including erythrocytes. The sequence of YSA1H contains a MutT sequence signature motif. A major proposed function of the MutT motif proteins is to eliminate toxic nucleotide metabolites from the cell. Hence the function of YSA1H might be to remove free ADP-ribose arising from NAD(+) and protein-bound poly- and mono-(ADP-ribose) turnover to prevent the occurrence of non-enzymic protein glycation. (+info)
A possible role for pyrophosphate in the coordination of cytosolic and plastidial carbon metabolism within the potato tuber.
The early stages of tuber development are characterized by cell division, high metabolic activity, and the predominance of invertase as the sucrose (Suc) cleaving activity. However, during the subsequent phase of starch accumulation the cleavage of Suc occurs primarily by the action of Suc synthase. The mechanism that is responsible for this switch in Suc cleaving activities is currently unknown. One striking difference between the invertase and Suc synthase mediated cleavage of Suc is the direct involvement of inorganic pyrophosphate (PPi) in the latter case. There is presently no convincing explanation of how the PPi required to support this process is generated in potato (Solanum tuberosum) tubers. The major site of PPi production in a maturing potato tubers is likely to be the reaction catalyzed by ADP-glucose pyrophosphorylase, the first committed step of starch biosynthesis in amyloplasts. We present data based on the analysis of the PPi levels in various transgenic plants altered in starch and Suc metabolism that support the hypothesis that PPi produced in the plastid is used to support cytosolic Suc breakdown and that PPi is an important coordinator of cytosolic and plastidial metabolism in potato tubers. (+info)
Legume embryos develop in a hypoxic environment.
Specific morphological and biochemical characteristics of seeds can cause oxygen deficiency within maternal and embryonic tissues. In this study, optical sensors were used to measure O(2) profiles across developing seeds of Vicia faba and Pisum sativum and developmental and environmental modulations of internal O(2) levels were studied. In addition, the metabolic state of developing embryos was analysed by monitoring adenylate energy charge, adenylate nucleotides and the levels of nucleotide sugars. Within the seed coat O(2) concentration decreased sharply to approximately 3% towards the inner border. Lowest O(2) levels were detected within the endospermal cavity between the seed coat and embryo. It is probable that low seed coat permeability provides an hypoxic environment for legume embryo development. The O(2) concentration in embryonic tissue changed during development with the lowest levels in the early stages. Measured in darkness, the levels were below 3%, but increased upon illumination indicating that photosynthesis significantly contributes to internal O(2) levels. Only in very young embryos were ATP levels and energy charge low. Otherwise they were maintained at a constant higher value. ADP-glucose and UDP-glucose did not show large fluctuations. Throughout embryo development fermentative activity did not play a major role. Obviously, specific mechanisms prevent seed tissues from becoming anoxic during development. The possible role of low oxygen on seed metabolism and on the control of seed development in legumes is discussed. (+info)
Starch synthesis and carbon partitioning in developing endosperm.
The biosynthesis of starch is the major determinant of yield in cereal grains. In this short review, attention is focused on the synthesis of the soluble substrate for starch synthesis, ADPglucose (ADPG). Consideration is given to the pathway of ADPG production, its subcellular compartmentation, and the role of metabolite transporters in mediating its delivery to the site of starch synthesis. As ADPG is an activated sugar, the dependence of its production on respiration, changes which occur during development, and the constraints which ATP production may place on carbon partitioning into different end-products are discussed. (+info)
A low-starch barley mutant, riso 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit.
To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Riso 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Riso 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene. (+info)
Identification and characterization of a critical region in the glycogen synthase from Escherichia coli.
The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding. (+info)
Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions.
Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism. (+info)