Jack Preiss

Regulation of the Amount of Starch in Plant Tissues by ADP Glucose Pyrophosphorylase

Starch, a major storage metabolite in plants, positively affects the agricultural yield of a number of crops. Its biosynthetic reactions use adenosine diphosphate glucose (ADPGlc) as a substrate; ADPGlc pyrophosphorylase, the enzyme involved in ADPGlc formation, is regulated by allosteric effectors. Evidence that this plastidial enzyme catalyzes a rate-limiting reaction in starch biosynthesis was derived by expression in plants of a gene that encodes a regulatory variant of this enzyme. Allosteric regulation was demonstrated to be the major physiological mechanism that controls starch biosynthesis. Thus, plant and bacterial systems for starch and glycogen biosynthesis are similar and distinct from yeast and mammalian systems, wherein glycogen synthase has been demonstrated to be the rate-limiting regulatory step.

From Glycogen to Amylopectin: A Model for the Biogenesis of the Plant Starch Granule

A major feature of the model we propose is that it gives us access to the third dimension of granule growth. The crystal lamella is a planar arrangement allowing for the three dimensional piling of glucan double helices (Figure 1Figure 1). The amorphous lamella on the other hand will not be planar but space-filling as can be predicted by the synthesis of phytoglycogen. At this stage the processing of phytoglycogen can lead to a variety of three dimensional structures that will allow for three dimensional extension of the amylopectin molecule. It is easy to understand how this is needed to accomodate regular concentric growth of the starch granule. Oostergetel and van Bruggen (1993) have very recently examined sections of potato starch granules by electron optical tomography and by cryo–electron diffraction. Their data imply a superhelical arrangement of both amorphous and crystalline lamellae. Moreover distinct superhelices are interlocked through their respective amorphous and crystalline lamellae to yield a tetragonal symmetry (Figure 3Figure 3). In this three dimensional arrangement, the double helical glucans are pointing in the axis of the superhelix towards the surface of the granule. This will of course allow for synthesis and growth of the crystals at the surface. This structure raises several questions with respect to biosynthesis, namely what determines the superhelical growth and how can this unidirectional growth account for concentric growth of the starch granule. We believe these questions can be presently addressed by our model. If we assume that the branching enzymes are setting the invariant amylopectin cluster size through their minimal catalytic requirements (see above), then once the first turn of the superhelix is synthesized the following turns will be dictated through this requirement. Concentric growth of the granule will call for synthesis of novel superhelices. These can be readily synthesized by allowing the amorphous lamella to fill vacant spaces between the growing superhelices. When sufficient space is available a novel superhelix will be made to grow by induced fit with the neighboring tetragonal organization. Debranching enzymes remain required at the surface to prevent glycogen synthesis and allow the trimming of the amorphous lamellae. The induced fit hypothesis for starch growth only requires the understanding of amylopectin cluster synthesis as proposed in our two dimensional model. Understanding how the first turn of the superhelices are generated will require further insight as to the priming events occurring at the granule core.Figure 3A Superhelical Model for the Three Dimensional Organization of Starch(A) The superhelical three dimensional organization of a section of the starch granule (based onOostergetel and van Bruggen 1993xOostergetel, G.T. and van Bruggen, E.F.J. Carbohydr. Polym. 1993; 21: 7–12Crossref | Scopus (138)See all ReferencesOostergetel and van Bruggen 1993). The top of the figure corresponds to the granules surface. The shaded areas correspond to the amorphous lamellae of the amylopectin molecules.(B) An enlargement of a single turn of the superhelix to display the double helices of the crystal lamellae. The shaded section would have overall structures similar to those shown for the amorphous lamellae in Figure 1Figure 1. Each superhelix is interlocked to neighboring superhelices to generate a tetragonal organization. We propose that vacant spaces are filled with amorphous material until sufficient room is available to yield a novel superhelix.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Branching of amylose by the branching isoenzymes of maize endosperm

Abstract A convenient, quantitative assay method of branching enzyme (BE) was devised with reduced amylose as the substrate. Using this assay, the properties of the purified branching isoenzymes from maize, BE I, IIa, and IIb, were studied. The method is based on determination of reducing power, by the modified Park-Johnson method, of the chains transferred by BE after they are released from the branched products with isoamylase. The optimum pH of the three enzymes is 7.5, and the optimum temperatures of BE I, IIa, and IIb are 33, 25, and 15–20°C, respectively. The specific activities are found to be the highest for BE I and the lowest for BE IIb, whereas in the conventional assay based on stimulation of unprimed phosphorylase activity, the specific activities are BE IIb > 11a > I. BE I has a lower Km (2.0 μM of the nonreducing terminal) for the reduced amylose of average chain-length ( cl ) 405 than BE IIa (10 μM) and IIb (11μM), and the enzyme shows a higher Km for reduced amyloses of smaller cl . Gel-permeation chromatograms on Sephadex G-75SF of the chains transferred from the reduced amylose indicate that initially the three isoenzymes produce chains of various sizes (dp ∼ 8 to > 200), and BE I preferentially transfers longer chains than BE IIa and IIb.

Differentiation of the Properties of the Branching Isozymes from Maize (Zea mays)

The multiple forms of branching enzyme (BE) from developing maize (Zea mays) endosperm were purified by modification of previous procedures such that amylase activity could be eliminated completely from the BE preparation. Three distinct assays for BE activity (phosphorylase a stimulation assay, BE linkage assay, and iodine stain assay) were used to characterize and differentiate the properties of the BE isoforms. This study presents the first evidence that the BE isoforms differ in their action on amylopectin. BEI had the highest activity in branching amylose, but its rate of branching amylopectin was less than 5% of that of branching amylose. Conversely, BEII isoforms had lower rates in branching amylose (about 9–12% of that of BEI) and had higher rates of branching amylopectin (about 6-fold) than BEI. The implication of these findings to the mechanism of amylopectin synthesis in vivo are discussed.

Biosynthesis of Starch and Its Regulation

The first in vitro demonstration of (1→4)-α-glucosidic linkage formation in plants occurred in 1940 when Hanes (1940) demonstrated the formation of an amylose product from glucose-1-P using potato extracts [reaction (1)].

ADP-Glucose Pyrophosphorylase: A Regulatory Enzyme for Plant Starch Synthesis

In plants, the synthesis of starch occurs by utilizing ADP-glucose as the glucosyl donor for the elongation of α-1,4-glucosidic chains. In photosynthetic bacteria the synthesis of glycogen follows a similar pathway. The first committed step in these pathways is the synthesis of ADP-glucose in a reaction catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). Generally, this enzyme is allosterically regulated by intermediates of the major carbon assimilatory pathway in the respective organism. In oxygenic photosynthesizers, ADPGlc PPase is mainly regulated by 3-phosphoglycerate (activator) and inorganic orthophosphate (inhibitor), interacting in four different patterns. Recent reports have shown that in higher plants, some of the enzymes could also be redox regulated. In eukaryotes, the enzyme is a heterotetramer comprised of two distinct subunits, a catalytic and a modulatory subunit. The latter has been proposed as related to variations in regulation of the enzyme in different plant tissues. Random and site-directed mutagenesis experiments of conserved amino acids revealed important residues for catalysis and regulation. Prediction of the ADPGlc PPase secondary structure suggests that it shares a common folding pattern to other sugar-nucleotide pyrophosphorylases, and they evolved from a common ancestor.

ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme for Bacterial Glycogen Synthesis

SUMMARY The accumulation of α-1,4-polyglucans is an important strategy to cope with transient starvation conditions in the environment. In bacteria and plants, the synthesis of glycogen and starch occurs by utilizing ADP-glucose as the glucosyl donor for elongation of the α-1,4-glucosidic chain. The main regulatory step takes place at the level of ADP-glucose synthesis, a reaction catalyzed by ADP-Glc pyrophosphorylase (PPase). Most of the ADP-Glc PPases are allosterically regulated by intermediates of the major carbon assimilatory pathway in the organism. Based on specificity for activator and inhibitor, classification of ADP-Glc PPases has been expanded into nine distinctive classes. According to predictions of the secondary structure of the ADP-Glc PPases, they seem to have a folding pattern common to other sugar nucleotide pyrophosphorylases. All the ADP-Glc PPases as well as other sugar nucleotide pyrophosphorylases appear to have evolved from a common ancestor, and later, ADP-Glc PPases developed specific regulatory properties, probably by addition of extra domains. Studies of different domains by construction of chimeric ADP-Glc PPases support this hypothesis. In addition to previous chemical modification experiments, the latest random and site-directed mutagenesis experiments with conserved amino acids revealed residues important for catalysis and regulation.

Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources.

SummaryThe primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.

Interaction of spinach leaf adenosine diphosphate glucose α-1,4-glucan α-4-glucosyl transferase and α-1,4-glucan, α-1,4-glucan-6-glycosyl transferase in synthesis of branched α-glucan

Abstract Chromatography of spinach leaf extracts on DEAE-cellulose resolves α-1,4-glucan, α-1,4-glucan 6-glycosyl transferase (branching enzyme) into two fractions. Branching enzyme fraction I which contains 10–20% of the total activity recovered from the DEAE-cellulose column is associated with the ADP-glucose α-1,4-glucan α-4-glucosyl transferase (α-glucan synthetase) fraction III. Separation of the branching enzyme I activity from the α-glucan synthetase III activity was achieved only by chromatography on an ADP-hexanolamine-Sepharose 4B column. The properties of the two branching enzyme fractions were very similar with respect to their activities toward potato amylose, amylopectin, and amyloses prepared with either rabbit muscle phosphorylase or spinach leaf α-glucan synthetase. Incubation of potato amylose with branching enzyme or incubation of phosphorylase or α-glucan synthetase with branching enzyme resulted in formation of glucan products resistant to complete hydrolysis by β-amylase (37–50%) and α-amylase (20–31%) but still quantitatively hydrolyzed by the combined action of pullulanase plus β-amylase. The only significant difference noted between the two branching enzyme activities is that fraction II has higher activity in citrate buffer than in other buffers in the pH range of 5.5–6.5 while fraction I has higher activity in bicine and phosphate buffers than with citrate buffer. Both branching enzyme fractions stimulate the previously described “unprimed activity” catalyzed by α-glucan synthetase fraction III about 11- to 14-fold. However, branching enzyme did not stimulate the primed activity. It was previously shown that the “unprimed reaction” was highly dependent on the presence of citrate and other anions. However, the K m of amylopectin and glycogen for α-glucan synthetase III which was found to be 0.53 mg/ml and 2.94 mg/ml, respectively, are decreased to 1.9 μ/ml and 0.86 μ/ml, respectively, in the presence of 0.5 m citrate. The levels at which primer are effective in the presence of 0.5 m citrate suggest that the “unprimed” synthetic activity previously reported may be due to the ability of the endogenous glucan associated with the α-glucan synthetase fraction to act as a primer when citrate or other anions are present. The stimulation of the “unprimed” activity by branching enzyme may then be explained by its catalysis of formation of an increased number of nonreducing chain ends in the growing glucan that are able to accept glucosyl residues from ADPglucose.

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Abstract Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A 1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d -glucose into the growing α- d -glucan and the synthesis of high molecular-weight polymers. However, the α- d -glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than II a.