Mirta N. Sivak

Biochemistry, Molecular Biology and Regulation of Starch Synthesis

This chapter reviews starch synthesis in higher plants, with special reference to the steps that can be manipulated to advantage by genetic manipulation. Thus, the enzymology and biochemistry of the various enzymes in the plant, algal and cyanobacterial systems will also be described as these are the potential sites for manipulation of both starch quantity and quality. Regulation of starch synthesis at the enzymatic and cellular levels will also be discussed with emphasis on its relevance to genetic engineering.

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.

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans.

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.

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, Cys7, Cys379, and Cys408, were substituted combinatorially by Ser. 5,5′-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys379 was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S0.5 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 Cys379 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 Cys379 is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu377 by Ala and Gln decreased Vmax more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu377 by Asp decreased Vmax only 57-fold indicating that the negative charge of Glu377 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, Ser374 and Gln383, 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.

Biochemical Evidence for the Role of the Waxy Protein from Pea (Pisum sativum L.) as a Granule-Bound Starch Synthase.

Proteins were solubilized from starch extracted from developing pea (Pisum sativum L.) embryos and chromatography of these proteins on a Mono-Q column separated two peaks of starch synthase activity. The major activity peak comprised more than 80% of the total activity. This fraction contained only the Waxy protein, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by staining for proteins or by immunoblot. A 77-kD polypeptide associated with the starch granules and presumed by others to be a starch synthase could not be detected in any of the active fractions. The native molecular weight of the solubilized starch synthase was 59,600 [plus or minus] 1700 as determined by sucrose density gradient. It is concluded that in pea seeds the Waxy protein and the starch synthase bound to the granule are the same protein.

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.

Progress in the genetic manipulation of crops aimed at changing starch structure and increasing starch accumulation

The starch content and its composition have important consequences for the yield of the harvested crop and the materials extracted from it. The functional properties of the foods or other processed materials derived from these crops are also affected by the structure and composition of the starch. Recently, genetic engineering has been used to produce plants with an elevated starch content, achieved by transforming the plant with a mutated bacterial gene coding for an ADPglucose pyrophosphorylase that is active in the presence of metabolites which inhibit the plant enzyme. Besides the practical implications of these results, this experiment provided direct evidence for the regulatory role of the ADPglucose pyrophosphorylase in starch synthesis. Other bacterial enzymes, such as glycogen synthase and branching enzyme, could be introduced in order to modify starch structure. However, a more elegant (but longer-term) approach would be to learn enough about the structure-function relationships of the plant enzymes so that the product of their action could be changed. To achieve this objective, much more will have to be learned about the enzymes involved in the biosynthesis of starch than is presently known. Here, the basic properties of starch and the current research approaches to understanding its biosynthesis are described, together with a perspective of how genetic manipulation of starch structure may be achieved.

(1 → 4)-α-d-Glucan synthesis by a chloroplastic phosphorylase isolated from spinach leaves in independent of added primer

Abstract The chloroplastic phosphorylase from spinach leaf was partially purified using ammonium sulfate fractionation and chromatography on DE-52 and Mono-Q columns. The enzyme was capable of synthesizing (1 → 4)-α- d -glucan in the absence of added glucan primer, with a lag in time that was shortened by increasing enzyme concentration or by adding bovine serum albumin and/or sodium citrate. The product of the unprimed activity formed a blue complex with iodine-iodide reagent (peak of absorption between 600 and 620 nm) that was insoluble in 5% trichloroacetic acid and in 1% (w/v) KCl in 75:25 methanol-water. Umprimed phosphorylase activity was greatly stimulated by citrate and less by other anions like malate and succinate. Addition of branching enzyme to the assay medium stimulated the unprimed reaction two-fold, or 42-fold if assayed in the presence of 0.1 m citrate. In the primed phosphorylase reaction, the presence of 0.8 m citrate decreased the K m for glycogen and amylopectin from 1.8 and 0.17 mg/mL to 0.12 and 0.055 mg/mL, respectively. The phosphorylase fraction (after f.p.l.c.) was found to contain 27 nmol of “anhydroglucose” per mg of protein. It is concluded that the chloroplast phosphorylase from spinach leaves in capable of synthesizing (1 → 4)-α- d -glucan in the absence of added glucan primer, possibly by elongating an endogenous primer that is associated with the enzyme in a non-covalent fashion.