Hanping Guan

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.

Isolation and characterization of the zSSIIa and zSSIIb starch synthase cDNA clones from maize endosperm

Two starch synthase clones, zSSIIa and zSSIIb, were isolated from a cDNA library constructed from W64A maize endosperm. zSSIIa and zSSIIb are 3124 and 2480 bp in length, and contain open reading frames of 732 and 698 amino acid residues, respectively. The deduced amino acid sequences of the two clones share 58.1% sequence identity. Amino acid sequence identity between the zSSIIa and zSSIIb clones and the starch synthase II clones of potato and pea ranges between 45 to 51%. The predicted amino acid sequence from each SSII cDNA contains the KXGGL consensus motif at the putative ADP-Glc binding site. Both clones also contain putative transit peptides followed by the VRAA(E)A motif, the consensus cleavage site located at the C-terminus of chloroplast transit peptides. The identity of the zSSIIa and zSSIIb clones as starch synthases was confirmed by expression of enzyme activity in Escherichia coli. Genomic DNA blot analysis revealed two copies of zSSIIa and a single copy of zSSIIb. zSSIIa was expressed predominantly in the endosperm, while transcripts for zSSIIb were detected mainly in the leaf at low abundance. These findings establish that the zSSIIa and zSSIIb genes are characteristically distinct from genes encoding granule-bound starch synthase I (Waxy protein) and starch synthase I.

Analysis of the active center of branching enzyme II from maize endosperm.

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2–4) of the four highly conserved regions (regions 1–4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Expression of Branching Enzyme I of Maize Endosperm in Escherichia coli

The gene encoding for mature branching enzyme (BE) I (BEI) of maize (Zea mays L.) endosperm has been expressed in Escherichia coli using the T7 promoter. The expressed BEI was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The recombinant enzyme showed properties very similar to those of BEI purified from developing maize endosperm with respect to branching amylose and amylopectin. This result confirmed our earlier report that maize endosperm BEI had a higher rate of branching amylose and a much lower rate (less than 10% of that of branching amylose) of branching amylopectin. This study also showed a great advantage in purifying BEI from the bacterial expression system rather than from developing maize endosperm. Most important, this study has established the system with which to study the structure-function relationships of the maize BEI using site-directed mutagenesis.

Influence of maize starch granule-associated protein on the rheological properties of starch pastes. Part II. Dynamic measurements of viscoelastic properties of starch pastes

The influence of granule-bound starch synthase (GBSS) on viscoelastic properties of gelatinized starch pastes was studied using a normal, a waxy null, and two GBSS-containing waxy mutant maize starches (waxy protein was synthesized with no GBSS activity). The four starches were isolated using the toluene method (isolation procedure I) (IP I) and were then further purified using an extended period of washing (isolation procedure II) (IP II). Dynamic rheological measurements of IP I gelatinized starches showed that storage moduli (G′) were higher in the two GBSS-containing waxy starches than in the waxy null mutant starch not containing GBSS. The total non-GBSS granule-associated proteins of the three waxy starches were similar. Further removal of granule-associated proteins by purification decreased the G′, with decreases higher in the two GBSS-containing waxy mutants than in the waxy null mutant starch. This was interpreted to be due to the removal of GBSS during purification of the former. High shear broke gelatinized IP I waxy and normal maize starches starch granule or remnant structure, which caused a decrease in G′. Cox–Merz plots showed that the elastic properties of the starch pastes were reduced by removal of starch granule-associated proteins.

Essential arginine residues in maize starch synthase IIa are involved in both ADP-glucose and primer binding

The arginine‐specific reagent phenylglyoxal inactivated the activity of maize starch synthase IIa (SSIIa), due to the modification of at least one arginine residue out of a possible 42. The addition of ADPGlc completely protected SSIIa from the inactivation, indicating that arginine may be involved in the interaction of this anionic substrate with SSIIa. However, site‐directed mutagenesis of the conserved Arg‐214 in SSIIa showed that this amino acid is important for apparent affinity of SSIIa for its primer (amylopectin and glycogen), as evidenced by a marked increase in the K m for primer upon substitution of this amino acid with no concomitant change in V max, K m for ADPGlc, or secondary structure. Therefore, Arg‐214 of SSIIa appears to play a role in its primer binding.

PROSPECTS FOR THE PRODUCTION OF CEREALS WITH IMPROVED STARCH PROPERTIES

The dominant pathway for the synthesis of starch involves three enzymes; ADPglucose pyrophosphorylase (ADPGlc PPase; EC 2.7.7. 27), which catalyzes the synthesis of ADPglucose; starch synthase (EC 2.4.1.21), which transfers the glucosyl portion of ADPglucose to a maltodextrin primer for synthesis and elongation of the α-1, 4 glucosyl chain and the branching enzyme (EC; 2.4.1.18) which transfers a portion of the elongated α-1, 4 glucosyl chain to form the α-1, 6 branch points present in amylopectin (and to small extent, in amylose). The physical properties of the starch in a plant are dependent on the properties and catalytic activities of the three enzymes mentioned above and alteration of the enzyme amounts and their properties will in turn, affect the properties of the starch synthesized. Recent results demonstrate that transformation of certain plants with a bacterial ADPGlc PPase gene can dramatically increase their starch content. It is quite possible that alteration of the proportion or amounts of branching enzyme and/or starch synthase would alter the structure and physical properties of the starch synthesized. To this end, the purified branching isoenzymes and soluble starch synthase isozymes are characterized to determine their specific roles in the synthesis of amylopectin. Preliminary experiments suggest that maize BE I isoenzyme is mainly involved in synthesis of the B chains of amylopectin while BE IIa and IIb are involved in the synthesis of the A chains.