Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K_m for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

The citrate-stimulated starch synthase of starchy maize kernels: Purification and properties

Chromatography of maize kernel extracts on DEAE-cellulose resolves two fractions of starch synthase activity, one of which (starch synthase 1) is capable of synthesizing α-glucan in the absence of exogenous primer and the presence of 0.5 m citrate (J. L. Ozbun, J. S. Hawker, and J. Preiss, Plant Physiol. (1971) 48, 765–769). This starch synthase has been purified 200-fold from developing kernels of normal maize, and shown to have no detectable activities of branching enzyme, amylase, pullulanase, phosphorylase, and D enzyme. The preparation, however, was not electrophoretically homogeneous. This preparation had a K_m value of 0.033 mm for ADPglucose in the presence of 0.5 m citrate. The reaction in the presence of citrate was stimulated 10-fold by the addition of excess purified branching enzyme. This stimulation is higher than those reported previously (C. D. Boyer and J. Preiss, Plant Physiol. (1979) 64, 1039–1042) but is consistent with the predicted effects of removal of amylase activity. The effects of salts other than citrate on activity in the absence of exogenous primer were small, but the stimulation could be restored by the addition of excess purified branching enzyme. Citrate increased the affinity of the enzyme for the endogenous primer present to such a level that no effect of exogenous primer on reaction rate could be observed in the presence of 0.5 m citrate. Analysis of the glucan/iodine complex and the enzymatic breakdown products patterns from the products of the starch synthase reaction indicates a high degree of linearity. The results obtained are discussed in relation to the biosynthesis of starch in vivo.     

Enzymes of starch metabolism in leaves and berries of Vitis vinifera

Leaves of Vitis vinifera L., cv. Cabernet Sauvignon contained 2.0 mg of starch per g fresh weight, whereas young green berries and maturing grape berries contained less than 0.03 mg of starch, despite the presence of abundant substrates (reducing sugars and sucrose) in berries for starch synthesis. the activities of several enzymes likely to be involved in starch synthesis were determined in extracts of berries and leaves. Fractionation procedures resulted in final recoverable ADPglucose-starch glucosyltransferase activity which was 2–3 times the activity measured in crude extracts of leaves. Compared to leaves, berries contained low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase. These enzymes increased only 2- to 3-fold from young to maturing berries. ADPglucose-starch glucosyltransferase activity in the absence of added primer was found in leaf extracts but not in berry extracts. The activities of UDP-glucose pyrophosphorylase, phosphorylase and amylase were comparable in both leaves and berries and increased 6- to 7-fold during berry development. The low activities of ADPglucose-starch glucosyltransferase and ADPglucose pyrophosphorylase probably account for the paucity of starch in grape berries.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

Combined action of Escherichia coli glycogen synthase and branching enzyme in the so-called "unprimed" polyglucoside synthesis.

Mutants of Escherichia coli which are unable to synthesize glycogen were used to study the so-called “unprimed” synthesis of glycogen. The glycogen synthase has been partially purified from these mutants. During the purification, attempts were made to separate the activity which requires the addition of an exogenous primer (primed activity) from the activity which does not require a primer but is highly dependent on the presence of some salts such as citrate and EDTA (unprimed activity). No separation between these two activities could be achieved but the results obtained by chromatography on DEAE-Sephadex indicate that there is a single form of glycogen synthase which is responsible for both unprimed and primed activity. The evidence that a single protein was necessary to catalyze these two reactions was given by the findings that mutants defective in glycogen synthase activity were unable to catalyze glucosyl transfer without added primer. At low concentration, the glycogen synthase purified from a branching enzyme negative mutant catalyzed the unprimed reaction at a slow rate even in presence of salts. A protein activator of this reaction was found in mutants lacking glycogen synthase but not in mutants lacking branching enzyme. The hypothesis that this activator is the branching enzyme itself was supported by the observation that it co-purified with the branching enzyme from a E. coli strain defective in glycogen synthase activity. EDTA or Triton X-100 increased the stimulation of the unprimed synthesis by the branching enzyme. The apparent affinity of the glycogen synthase for glycogen was increased twofold in the presence of EDTA but the branching enzyme further increased the effect of EDTA. The combined action of the glycogen synthase and the branching enzyme on the endogenous glucan associated with the synthase may account for the unprimed activity observed in vitro.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Possible common origin of glucosyltransferases in Oscillatoria princeps

Gel electrophoresis of the glucosyltransferases of the blue-green alga, Oscillatoria princeps, followed by immunodiffusion against anti-phosphorylase rabbit serum, showed cross-reactions of the two phosphorylase isozymes and the two synthetase isozymes of the alga. Weak cross-reactions were also obtained with the branching isozymes. Apparent extensive similarities in the structure of the phosphorylases and the synthetases were indicated. However, only partial structural similarities between the two groups of α-1,4-glucosidic bond formers and the branching isozymes exist as indicated by the weak immunological reactions obtained and the formation of “spurs” on the immunoprecipitin lines. If the synthesis of α-1,4-glucosidic linkages and the formation of α-1,6 cross linkages were at one time due to the bifunctional action of a single catalytic protein, then the separation of these two enzymatic activities took place prior to the derivation of the synthetases from the phosphorylases.     

Gene dosage at the amylose-extender locus of maize: Effects on the levels of starch branching enzymes

Soluble starch branching enzymes and starch synthases from maize kernels of differing dosage of the ae locus were purified by DEAE-cellulose chromatography. A near-linear relationship between increasing dosage of the dominate amylose-extender allele (Ae) and branching enzyme IIb activity was found. In contrast, levels and properties of branching enzymes I and IIa, as well as the citrate-stimulated and primer-requiring starch synthases, remained unchanged. The near-linear increase in branching enzyme IIb activity with increasing doses of the Ae allele is consistent with the hypothesis that ae is the structural gene coding for branching enzyme IIb.Paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick. This work was performed as part of NJAES Projects 12442 and 12201 (NE-124), supported by the New Jersey Agricultural Experiment Station, Regional Research Funds, NSF Grant PCM 78-16127, and funds from Corn Refiners Association, Inc.     

Carbohydrate metabolism in Musa paradisiaca

Considerable variations exist in the content of glucose, fructose, sucrose, starch and protein and in the activities of enzymes involved in carbohydrate metabolism between different parts of the banana plant (Musa paradisiaca). Sucrose synthetase is present in the highest concentration in rootstock and fruit pulp, and sucrose phosphate synthetase in the pseudostem. The highest ratio of the activity of sucrose phosphate synthetase to sucrose synthetase is found in leaves. Acid invertase is present in leaves, leaf-sheath and fruit pulp and is not demonstrable in rootstock and pseudostem. Neutral invertase activity is high in pseudostem and leaf-sheath. Starch phosphorylase is largely concentrated in fruit pulp and rootstock. The maximum activity of ATP:d-phosphoglucose (ADPG) pyrophosphorylase is found in rootstock. β-Amylase is not demonstrable in rootstock and is largely concentrated in leaf-sheath. Hexokinase is most active in rootstock and the lowest in leaves. Acid phosphatase and alkaline phosphatase activity is highest in fruit pulp and pseudostem. Glucosephosphate isomerase is most active in the rootstock and lowest in the leaves.     

Characterization of substrate and product specificity of the purified recombinant glycogen branching enzyme of Rhodothermus obamensis

Background

Glycogen and starch branching enzymes catalyze the formation of α(1 → 6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1 → 4) linkage and transfer in α(1 → 6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch.

Methods

The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure.

Results

A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3–8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30–70 nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family.

Conclusions

RoBE is able to transfer α(1 → 4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1 → 4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family.

General significance

This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro.     

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

The requirement for a primer in the in vitro synthesis of polysaccharide by sweet-corn (1 → 4)-α-d-glucan syntrase

A mixture of (1 → 4)-α-d-glucan synthases was partially purified from sweet corn. The synthesis of polysaccharide from ADP-d-glucose by the enzyme preparation was dependent on added carbohydrate primer in solutions of low ionic strength, but displayed the phenomenon of being apparently primer-independent at high ionic strength in citrate buffer. This phenomenon was further investigated; treatment of the enzyme preparation with immobilized amylases led to the abolition of the apparently unprimed synthesis. The amylase-treated preparation then showed a normal dependence on (1 → 4)-α-d-glucan primer, branched primers being the most effective. The affinity of the enzyme for a branched primer appeared to be enhanced in the presence of citrate. The polysaccharide product of the unprimed reaction was glycogen-like, having an average chain-length of 14. These studies suggest that the phenomenon of unprimed synthesis in “high salt” is explicable in terms of an enhanced affinity of the enzyme for traces of primer in the enzyme preparation, and not to a “de novo” synthesis of polysaccharide, that occurs in the absence of a primer.     

Molecular weight of starch synthetase from Oryza sativa leaves

Soluble ADP-glucose: α-1,4-glucan-4-glucosyltransferase with primed activity was extracted from rice leaves and purified by (NH₄)₂SO₄ fractionation, gradient elution on DEAE-cellulose and finally by Sephadex G200 gel filtration or amylopectin-cellulose chromatography. The purified enzyme was essentially homogeneous electrophoretically, but exhibited two peaks corresponding to MW of 22 000 and 67 000 on Sephadex G200 chromatography and five distinct bands on sodium dodecyl sulfate gel electrophoresis with MW of 11·5, 20, 35, 50 and 68 × 10³.     

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

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 IIa.     

Immunological comparison of the starch branching enzymes from potato tubers and maize kernels

Starch branching enzyme was purified from potato (Solanum tuberosum L.) tubers as a single species of 79 kilodaltons and specific antibodies were prepared against both the native enzyme and against the gel-purified, denatured enzyme. The activity of potato branching enzyme could only be neutralized by antinative potato branching enzyme, whereas both types of antibodies reacted with denatured potato branching enzyme. Starch branching enzymes were also isolated from maize (Zea mays L.) kernels. All of the denatured forms of the maize enzyme reacted with antidenatured potato branching enzyme, whereas recognition by antinative potato branching enzyme was limited to maize branching enzymes I and IIb. Antibodies directed against the denatured potato enzyme were unable to neutralize the activity of any of the maize branching enzymes. Antinative potato branching enzyme fully inhibited the activity of maize branching enzyme I; the neutralized maize enzyme was identified as a 82 kilodalton protein. It is concluded that potato branching enzyme (M_r = 79,000) shares a high degree of similarity with maize branching enzyme I (M_r = 82,000), in the native as well as the denatured form. Cross-reactivity between potato branching enzyme and the other forms of maize branching enzyme was observed only after denaturation, which suggests mutual sequence similarities between these species.