CHEMICAL BIOLOGY

Starch Biosynthesis In Plants

 

Jack Preiss, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan

doi: 10.1002/9780470048672.wecb191

 

Starch is a major storage compound in plants that is present both in leaves and in storage tissues. Biochemical and molecular biological data show that ADP-glucose is the glucosyl donor for plant starch synthesis, and its synthesis is catalyzed by ADP-glucose pyrophosphorylase. Subsequently, starch synthases catalyze the transfer of the glucosyl residue from ADP-glucose to the oligosaccharide chains of the starch components amylose and amylopectin to form new α-1,4-glucosidic residues. After elongation of these α-1,4-glucosidic chains, the branching enzyme catalyzes a cleavage of the elongated chain and transfers the cleaved portion of the oligosaccharide chain to either another region in the amylopectin molecule or to a new amylopectin and forms a new α-1,6-glucosidic linkage. Amylose synthesis is catalyzed by the granule-bound starch synthase. Regulation of starch synthesis occurs at the ADP-glucose pyrophosphorylase step. The enzyme from higher plants, green algae, and cyanobacteria is activated allosterically by 3-phosphoglycerate and inhibited by inorganic phosphate. Isolation of mutants and control analyses indicate that the allosteric activation and inhibition are of physiological and functional importance in the regulation of starch synthesis. Furthermore, evidence indicates that ADP-glucose pyrophosphorylases can also be regulated by a redox mechanism. The current knowledge of the enzyme structures and critical amino acids necessary for substrate binding, allosteric effector binding, regulation, and catalysis for the ADP-glucose pyrophosphorylase is reviewed.

 

The enzymatic reactions involved in starch synthesis in higher plants and algae and in glycogen synthesis in cyanobacteria are reviewed in this article. Regulation of α-1,4 and α-1,6 glucan synthesis at the enzymic level is discussed, and mutants that cause specific enzymatic deficiencies that affect starch structure are also reviewed. Recent reviews on starch synthesis have been published and are noted (1-6).

 

Localization of Starch in Plants

Starch is present in every type of tissue, such as leaves, fruit, pollen grains, roots, shoots, and stems. Formation of starch granules occurs in the chloroplast during exposure of leaves to bright light because of carbon uptake during photosynthesis. Loss of leaf starch occurs in low light or in long dark periods of 24-48 hours where it is degraded to products that are used for sucrose synthesis. Arabidopsis thaliana mutants that cannot synthesize starch grow as well as wild type when placed in continuous light as they can synthesize sucrose (7). However, if grown in a day-night regime, the growth rate is dramatically decreased because starch is required for sucrose synthesis at night. Thus, leaf starch synthesis and use is a dynamic process with diurnal fluctuations.

Starch synthesis occurs during development and maturation of plant storage organs. During sprouting or germination of the seed or tuber or in fruit ripening, the reserved starch is degraded, and its metabolites are sources for carbon and energy. Thus, reserved starch degradation and synthesis are time-separated events. Cereal grain starch-storage tissue is the endosperm, with starch granules located in the amyloplasts. Reserve starch content varies from 65% to 90% of the total dry weight.

The starch granule contains two polysaccharide types: amylose, which is a linear polymer, and amylopectin, which is a branched polymer. Amylose is composed of linear chains of about 800 to 22,000 α-D-glucopyranosyl units in (α-1,4) linkage (molecular size ~ 1.36 x 105 to 3.5 x 106). Amylose molecules may be slightly branched (branch chain, 170 to 500 glucosyl units). Amylopectin, which comprises about 70-80% of the starch granule, has 4-5% of the glucosidic linkages as branch points.

The accepted models of proposed amylopectin structures, which are known as cluster models, are those of Robin et al. (8), Manners and Matheson (9), and Hizukuri (10). Reviews that detail chemical and physical properties of starch are given by Morrison and Karkalis (11) and Hizukuri (12). Figure 1 shows the proposed cluster structure.

 

 

Figure 1. Cluster model of amylopectin (10). A indicates A chains. A chains have no other oligosaccharide chains attached to them. B indicates B chains that are defined as having either one or more oligosaccharide chains (either A or B) attached to them. The C chain, which is not labeled, has the only reducing end group Ø in the polysaccharide. B3 chains are longer than B2 chains, which are longer than the B1 chains. The B2, B3, and B4 (not shown) chains extend into 2, 3 and 4 cluster regions, respectively. The average chain lengths are B1, 19; B2, 41; B3, 169; and B4, 109. For the A chains, the shortest chain length is about 13.

 

Starch Synthesis Reactions in Plants and Algae and Glycogen Synthesis in Cyanobacteria

ADP-glucose is used for synthesis of α-1,4 glucosidic linkages in amylose and amylopectin. Its synthesis is catalyzed by ADP-glucose pyrophosphorylase (reaction 1, E.C. 2.7.7.27; ATP: α-D-glucose-1-phosphate adenylyltransferase).

Synthesis of the 1,4-α-D-glucosidic bond in starch is catalyzed by starch synthase (E.C. 2.4.1.21; ADP-glucose: 1,4-α-D-glucan 4-α-glucosyltransferase; reaction 2) and was first reported by Leloir (13,14). Similar reactions are seen for glycogen synthesis in bacteria and cyanobacteria (15), and the enzyme is termed glycogen synthase (also E.C. 2.4.1.21).

Synthesis of the 1,6-α-D-glucose bonds in amylopectin is catalyzed by a branching enzyme [E.C. 2.4.1.18; 1,4-α-D-glucan 6-α-(1,4-α-glucano)-transferase; reaction 3]. Amylopectin contains longer chains (about 20-24 glucosyl units) and has less branching (~5% of the glucosidic linkages are α-1, 6) than animal or bacterial glycogen (10-13 glucosyl units and 10% of linkages, α-1, 6).

Isozymes of plant starch synthases (2, 16-20) and branching enzymes (2, 16, 21-26) have been characterized. Presumably, they play distinct roles in amylopectin and amylose synthesis. Also, plant and Chlamydomonas reinhardtii granule-bound starch synthases (27-33) are responsible for synthesis of amylose. Mutants defective in this enzyme are known as waxy mutants and contain starch granules that have little or no amylose.

 

A debranching enzyme called isoamylase is involved in synthesis of the starch granule and its polysaccharide components (34-37). Mutant plants deficient in isoamylase activity accumulate a soluble a-glucan designated phytoglycogen (3, 37, 38) and little starch.

 

Properties of the Plant 1,4-α-Glucan Synthesizing Enzymes

ADP-glucose pyrophosphorylase: kinetic properties

The ADP-glucose pyrophosphorylases (ADP-Glc PPase) of higher plants, green algae, and the cyanobacteria are activated allosterically by 3-phosphoglycerate (3-PGA) and inhibited by inorganic phosphate (Pi). These effects are important in regulation of starch synthesis. Both potato tuber (2, 39-49) and spinach leaf ADP-Glc PPases (50-52) have been studied in detail with respect to kinetic properties. The kinetic and regulatory properties of the ADP-Glc PPases of several leaf extracts are similar to those of the spinach leaf enzyme (50). The results obtained with potato tuber and spinach leaf enzyme are summarized in Table 1.

 

Table 1. Kinetic constants of ADP-Glc PPases from Spinach leaf and potato tuber. S0.5 is the concentration of substrate ATP or Glc-1-P required to attain 50% of Vmax. A0.5 is the concentration of activator 3PGA required for 50% of maximal activation. I0.5 is the concentration of inhibitor need for 50% inhibition

 

Source

Effector/substrate

S0.5/A0.5/I0.5 (mM)

Hill constant n

Activation-fold

Reference

Spinach leaf

3-PGA

0.051

1.0

20

50

 

Pi

0.045

1.1

 

 

 

Pi (+3-PGA, 1 mM)

0.97

3.7

 

 

 

ATP

0.38

0.9

 

 

 

ATP(+3-PGA)

0.062

0.9

 

 

 

Glc-1-P

0.12

0.9

 

 

 

Glc-1-P(+3-PGA)

0.035

1.0

 

 

Potato tuber

3-PGA

0.16

1.0

30

42, 44

 

Pi (-3-PGA)

0.04

NR

 

 

 

Pi (+3-PGA, 3 mM)

0.63

NR

 

 

 

ATP (+3-PGA)

0.076

1.6

 

 

 

Glc-1-P (+3-PGA)

0.057

1.1

 

 

 

Quaternary structure

Bacterial ADP-Glc PPases are homotetrameric in structure (53). The catalytic and allosteric sites are on each subunit. In plants and green algae, however, the ADP-Glc PPases have been shown to be heterotetramers with two homologous subunits α2β2 (5, 52-54) that have different molecular sizes. The small subunits are 50-54 kDa, with catalytic activity. The large subunit, which is about 51-60 kDa, is the regulatory subunit. The large (regulatory) subunit modulates the sensitivity of the small subunit toward allosteric effectors via large-subunit/small-subunit interactions (45). However, recent results indicate that some large subunits, particularly those in the leaf (55), also have catalytic activity.

ADP-Glc PPase from potato tuber is composed of two different subunits, each of 50 and 51 kilodaltons, with α2β2 heterotetrameric-subunit structure. The potato tuber ADP-Glc PPase small subunit can be expressed as a homotetramer and is active in presence of high concentrations of the activator 3-PGA. The small subunit of many higher plant ADP-Glc PPases is highly conserved among plants with 85-95% identity (54). The homotetrameric potato enzyme that is composed exclusively of small subunits has a lower apparent affinity (A0.5 = 2.4 mM) for the activator 3-PGA than the heterotetramer (A0.5 = 0.16 mM), which is more sensitive to the inhibitor Pi (I0.5 = 0.08 mM in the presence of 3-mM 3-PGA) as compared with the heterotetramer (I0.5 = 0.63 mM) (42). The large subunit of the potato greatly increases the affinity of the small (catalytic) subunit for 3-PGA and lowers the affinity for the inhibitor Pi (41, 42).

In plants, only one conserved small (catalytic) subunit and several large (regulatory) subunits are distributed in different parts of the plant (56, 57). This finding is of physiological significance as expression of different large subunits in different plant tissues may confer distinct allosteric properties to the ADP-Glc PPase needed for the plant tissue's distinct need for starch.

It has been shown with Arabidopsis ADP-Glc PPase that coexpression of its small-subunit APS1 with the different Arabidopsis large subunits, which include ApL1, ApL2, ApL3, and APL4, resulted in heterotetramers with different regulatory and kinetic properties (56) (Table 2 and Table 3). The heterotetramer of the small-subunit APS1 with ApL1, which is the predominant leaf large subunit (56), had the highest sensitivity to the allosteric effectors 3-PGA and Pi as well as the highest apparent affinity for the substrates ATP and Glc-1-P. The het- erotetrameric pairs of APS1 with either APL3 or APL4, which are large subunits prevalent in sink or storage tissues (57), had intermediate sensitivity to the allosteric effectors and intermediate affinity for the substrates ATP and Glc-1-P (57). APL2 also present mainly in sink tissues had low affinity for either 3-PGA or Pi (56). Thus, differences on the regulatory properties conferred by the Arabidopsis large subunits were found in vitro. Differences noted for source and sink large-subunit proteins strongly suggests that starch synthesis is modulated in a tissue-specific manner in response to 3-PGA and Pi, as well as to the substrate levels. APS1 and ApL1 would be regulated finely in source tissues by both effectors and substrates, whereas in sink tissues, the hetrotetramers of APS1 with APL2, APL3, or APL4 with lower sensitivity to effectors, and substrates would be controlled more by the supply of substrates.

Based on mRNA expression, ApS1 is the main small subunit or catalytic isoform responsible for ADP-Glc PPase activity in all tissues of the plant. ApL1 is the main large subunit in source tissues, whereas ApL3 and ApL4 are the main isoforms present in sink tissues. It was also found that sugar regulation of ADP-Glc PPase genes was restricted to ApL3 and ApL4 in leaves (57). Sucrose induction of ApL3 and ApL4 transcription in leaves allowed formation of heterotetramers less sensitive to the allosteric effectors, which resembles the situation in sink tissues.

 

Table 2. Kinetic parameters for the 3-PGA of A. thaliana recombinant ADP-Glc PPase in the synthesis direction (56)

 

 

Control

 

0.2 mM Pi

 

2 mM Pi

 

3-PGA A0.5 mM

nH

3-PGA A0.5 mM

nH

3-PGA A0.5 mM

nH

APS1

5.7 ± 0.5

1.6

13.9 ± 1.8

2.3

ND

 

APS1/APL1

0.018 ± 0.004

0.8

0.094 ± 0.002

1.4

1.6 ± 0.1

2.9

APS1/APL2

0.87 ± 0.11

0.9

1.54 ± 0.27

1.3

10.5 ± 1.6

1.5

APS1/APL3

0.34 ± 0.09

0.8

0.70 ± 0.1

1.4

2.7 ± 0.1

2.7

APS1/APL4

0.16 ± 0.03

0.8

0.46 ± 0.1

1.2

1.7 ± 0.1

1.5

ND, not determined.

APS1 designates Arabidopsis thaliana small subunit 1, APL1, APL2, APL3, and APL4 are large subunits 1, 2, 3, and 4, respectively from A. thaliana.

 

Table 3. Arabidopsis ADP-Glc PPase: Kinetic parameters for the substrates in the synthesis direction (56)

 

 

ATP

 

Glc-l-P

 

S 0.5 mM

nH

S 0.5 mM

nH

APS1

0.402 ± 0.04

1.5

0.076 ± 0.018

0.9

APS1/APL1

0.067 ± 0.008

1.0

0.019 ± 0.001

1.0

APS1/APL2

0.575 ± 0.03

1.6

0.085 ± 0.014

0.9

APS1/APL3

0.094 ± 0.008

1.4

0.052 ± 0.007

1.0

APS1/APL4

0.118 ± 0.01

1.2

0.060 ± 0.008

0.9

20 mM of 3-PGA was used for the APS1 enzyme in determining the ATP and the Glc-1-P kinetic constants. For the APS1/APL1 kinetic study, 0.1 mM of 3PGA was used. 4mM of 3PGA was used for the APS1/APL2, 2mM of 3PGA was used for the APS1/APL3, and 1 mM of 3PGA was used for the APS1/APL4 kinetic studies. These concentrations were five times the 3-PGA A0.5 of each enzyme, respectively.

 

Relationship between the small and large subunits

The similarity between the small and large subunits (~50 to 60% identity) suggests a common origin (54). In both sink and source tissues, the small subunit has catalytic activity, whereas catalytic activity is only observed for the large subunits that may reside in the leaf and not in the sink large subunits (55). Gene duplication and divergence probably has led to different and functional catalytic and regulatory roles for the subunits. The ancestor of small and large subunits possibly is a bacterial subunit that has both catalytic as well as regulatory function in the same subunit. This suggestion is supported by the similarity between the two plant subunits with many active bacterial ADP-Glc PPases (5, 53).

The large subunit from the potato (Solanum tuberosum L.) tuber ADP-Glc PPase was shown to bind substrates (44). Therefore, the plant heterotetramer, and bacterial homotetramers bind four ADP- [14 C] glucose molecules (44, 58). It can be postulated that the large subunit maintained its structure needed for binding of substrate, but catalytic ability was eliminated by mutations of essential residues. To test this hypothesis, it was attempted to create a large subunit with significant catalytic activity by mutating as few residues as possible (49).

Thus, sequence alignments of ADP-Glc PPase large and small subunits with reported activity were compared to identify of critical missing residues for catalytic activity in the large subunit (49). The subset of the residues absent in the large subunit was of particular interest. In the large subunit of potato tuber, Lys44 and Thr54 were selected as the best candidates to study because the homologous residues Arg33 and Lys43 in the small (catalytic) subunit were conserved completely in the active bacterial and plant catalytic subunits. Moreover, Lys44 and Thr54 are in a highly conserved region of ADP-Glc PPases (Table 4).

The modulatory large subunits Lys44 and Thr54 were mutagenized to Arg44 and Lys54, respectively. The mutant, LargeK44R/T54K was expressed in the absence of the small subunit, and it had no activity. Possibly the large subunit cannot form a stable tetramer in absence of the small subunit as observed earlier with the Arabidopsis enzyme (56). Because a wild-type small subunit has intrinsic activity, the activity of the large-subunit mutants cannot be tested when coexpressed. Thus, the large-subunit mutants were coexpressed with inactive small-subunit D145N, in which the catalytic residueAsp145 was mutated (59), and small subunit activity was reduced by more than three orders of magnitude (Table 5). Coexpression of the large-subunit double mutant K44R/T54K with smallD145N generated an enzyme that had 10% and 18% of the wild-type enzyme in the ADP-glucose synthetic and pyrophos-phorolytic directions, respectively (Table 5). Single mutations of K44R or T54K generated enzymes with no significant activity. The combination of both mutations in the large subunit (smallD145NlargeK44R/T54K) gave the most dramatic effect (Table 5). Therefore, it was concluded that the two residues Arg44 and Lys54 are needed for restoring catalytic activity to the large subunit. Replacement of the homologous two residues with Lys and Thr in the small subunit (by mutations R33K and K43T) decreased the activity by one and two orders of magnitude, respectively, in both directions, which confirms the hypothesis (Table 5) (49). The mutant enzymes were still activated by 3-PGA and inhibited by orthophosphate (Pi). The wild-type (wt) enzyme and smallD145NlargeK44R/T54K had very similar kinetic properties, which indicated that the substrate site domain has been conserved. The apparent affinities for the substrates and the allosteric properties of small subunit D145NlargeK44R/T54K resembled those of the wild type (49). The new form has a similar sensitivity to Pi inhibition, and the activator-inhibitor interaction is the same. The large subunit restored enzyme activity because the two mutations provide evidence that the large and small subunits are derived from the same ancestor. The smallD145NlargeK44R/T54K mutant was disrupted in each subunit at their Glc-1-P site, and their kinetic properties were compared. With wt enzyme replacement of Lys198 in the small subunit of the wild-type enzyme, decreased the Glc-1-P affinity was observed; disruption of the homologous residue Lys213 in the large subunit had much less effect on the affinity (59). In smallD145NlargeK44R/T54K, the K213R mutation of a large subunit severely decreased the apparent affinity for Glc-1-P, whereas the K198R mutation on the small subunit did not indicate the large subunit double mutant, and not smallD145N was the catalytic subunit. In the wild-type enzyme, Lys213 does not seem to play an important role, but smallD145NlargeK44R/T54K recovered its ancestral ability for the enzyme to have a low physiological Km for Glc-1-P. Previous results showed that in wild type, Asp145 of the small subunit is essential for catalysis, but homologous Asp160 in the large subunit is not essential for catalysis (59). In addition, mutation of D160 to N or E in the active large subunit, LK44R/T54K abolished activity. This result confirms that catalysis of smallD145Nlarge K44R/T54K does occur in the large subunit.

A comparative model of LK44R/T54K shows the predicted role of Arg44 and Lys54 (Fig. 2). In the model, Asp160, which is homologous to the catalytic Asp145 in the small subunit and catalytic Asp142 in the E. coli ADP-Glc PPase (59, 60), interacts with Lys54. This type of interaction (Lys54-Asp160) has also been observed in crystal structures of enzymes that catalyze similar reactions, such as dTDP-glucose pyrophos-phorylase (dTDP-Glc PPase) and UDPN-N-acetyl-glucosamine pyrophosphorylase (UDPGlcNAc PPase). It is postulated to be important for catalysis by orienting the aspartate residue correctly (61-63). Lys54 interacts with the oxygen that bridges the α- and β-phosphates as it has been observed in the crystal structure of E. coli dTDP-Glc PPase (63). The interaction may neutralize a negative charge density to stablize the transition state and make PPi a better leaving group. Arg44 interacts in the model with the β- and γ-phosphates of ATP, which correspond to the PPi byproduct (Fig. 2). Likewise, Arg15 in the E. coli dTDP-Glc PPase was postulated to contribute to the departure of PPi (61), and kinetic data agreed with interaction of PPi with Arg44 in the model. A Lys44, in both the catalytic large subunit mutant and the small subunit, decreased the apparent affinity for PPi at least 20-fold (49). In a wild-type large subunit, Lys44 and Thr54 cannot interact as Arg44 and Lys54 (Fig. 2).

 

Table 4. Sequence comparison of the potato tuber large subunit (large wt) with ADP-Glc PPases shown to be enzymatically active (49)

 

Residues that are 100% conserved are in red and the ones conserved in the small subunit (small wt) but not in the large subunit are in blue.

 

Table 5. Activity of potato tuber small (catalytic) and large (regulatory) subunit ADP-Glc PPase mutants (49)

 

Small (catalytic)

Subunits

Large (regulatory)

Units/mg

ADP-glucose synthesis

ATP synthesis

WT

WT

32 ± 1

49 ± 2

D145N

WT

0.017 ± 0.001

0.037 ± 0.002

D145N

K44R

0.031 ± 0.001

0.033 ± 0.002

D145N

T54K

0.92 ± 0.08

0.56 ± 0.03

D145N

K44R/T54K

3.2 ± 0.2

9.0 ± 0.7

R33K

WT

3.6 ± 0.2

4.1 ± 0.1

K43T

WT

0.32 ± 0.1

0.28 ± 0.01

The enzymes activities of purified coexpressed small and large subunits were measured for ADP-glucose synthetic activity or ADP-glucose pyrophosphorolytic (ATP synthesis) activity. For ADP-glucose synthesis, 4mM of 3-PGA (activator), 2mM of ATP, and 0.5 mM of Glc-1-P were used. For pyrophosphorolysis, 4mM of 3-PGA, mM of ADP-Glc, and 1.4 mM of PPi were used.

 

 

Figure 2. Involvement of large (regulatory) subunit mutant K44 R/T54K in ADP-Glc PPase catalysis. The WT and double-mutant large subunits were modeled based on the dTDP-Glc PPase and UDP-UDPGlcNAc PPase crystal structures as indicated in the text. Portions of ADP-Glc PPase residues 31-73 and 131-136 are shown. The deoxyribose triphosphate portion common to dTTP and ATP is modeled with Mg+2 as a black sphere. The dotted lines depict hydrogen bonds.

 

Phylogenetic analysis of the large and small subunits

A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits (Fig. 3). The tree shows that plant small and large subunits can be divided into two and four distinct groups, respectively (49). The two main groups of small subunits are from dicot and monocot plants, whereas large-subunit groups correlate better with their documented tissue expression. The first large-subunit group, which is termed group 1, is generally expressed in photosynthetic tissues (49) and comprises large subunits from dicots and monocots. These subunits have been shown recently to have catalytic activity and have in their sequences Arg and Lys in the equivalent residues of 102 and 112 of A. thaliana large subunit, APL1. Group 2 displays a broader expression pattern, whereas groups 3 and 4 are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group 3 are only from dicot plants, whereas group 4 includes seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation (49). These subunits are probably inactive in catalytic activity as they are lacking Arg and Lys in the homologous residues observed in A. thaliana APLI and APL2.

 

 

Figure 3. Phylogenetic relationship of ADP-Glc PPases from photosynthetic eukaryotes (49). All ADP-Glc PPases are from angiosperms because no complete sequences are available from gymnosperms. Dotted lines separate large (L) from small (S) subunits. The cyanobacterial ADP-Glc PPase from Anabaena is included as an outgroup. The black arrow points to the potato tuber L subunit (sequence 66) analyzed in this study. The general topology of the tree has strong support from a maximum-likelihood method indicated in Supplemental Fig. (1) in Reference 49. The brackets show the residues conserved in each group in the residues that correspond to Lys44 (first bracket) and Thr54 (second bracket) in the potato tuber L subunit. The scale on the bottom left represents the number of substitutions per site.

 

Crystal structure of potato tuber ADP-Glc PPase

The crystal structure of potato tuber homotetrameric small (catalytic) subunit ADP-Glc PPase has been determined to 2.1A° resolution (48). The structures of the enzyme in complex with ATP and ADP-Glc were determined to 2.6 and 2.2 A° resolution, respectively. Ammonium sulfate was used in the crystallization process and was found tightly bound to the crystalline enzyme. It was also shown that the small-subunit homotetrameric potato tuber ADP-Glc PPase was also inhibited by inorganic sulfate with the I0.5 value of 2.8 mM in the presence of 6-mM 3-PGA (48). Sulfate is considered as an analog of phosphate, which is the allosteric inhibitor of plant ADP-Glc PPases. Thus, the atomic resolution structure of the ADP-Glc PPase probably presents a conformation of the allosteric enzyme in its inhibited state. The crystal structure of the potato tuber ADP-Glc PPase (48) allows one to determine the location of the activator and substrate sites in the three-dimensional structure and their relation to the catalytic residue Asp145. The structure also provides insights into the mechanism of allosteric regulation.

 

Supporting data for the physiological importance of regulation of ADP-glucose pyrophosphorylase

The reaction catalyzed by plant ADP-Glc PPases is activated by 3-P-glycerate and inhibited by orthophosphate; it is an important step for regulation of starch synthesis in higher plants as well as in the cyanobacteria (see reviews in References 5 and 53). In addition, it has been shown that in higher plants the enzyme activity can also be regulated by its redox state (46, 47) Considerable experimental evidence is available to support the concept that ADP-Glc PPase is an important regulatory enzyme for the synthesis of plant starch. A class of C. reinhardtii starch-deficient mutants have been isolated and shown to contain an ADP-Glc PPase not activated by 3-PGA (64, 65). Evidence for the allosteric regulation by ADP-Glc PPase being pertinent in vivo has also been obtained with A. thaliana (66, 67). Mutant TL25 lacked both subunits and accumulated less than 2% of the limits of detection of starch observed in the normal plant (66), which would indicate that starch synthesis is almost completely dependent on the synthesis of ADP-Glc. Mutant TL 46 is also starch deficient, and it lacked the regulatory 54-kDa subunit (67). The mutant had only 7% of the wild-type ADP-Glc PPase activity, and subsequently (68) it was shown that in optimal photosynthesis the starch synthetic rate of the mutant was only 9% of that of the wild type. At low light, starch synthesis in TL 46 was only 26% of the wild-type rate. These observations support the idea that regulation of ADP-Glc PPase by 3-PGA and Pi is physiologically pertinent. A maize endosperm mutant ADP-Glc PPase is less sensitive to inhibition by Pi than the wild-type enzyme (69), which had 15% more dry weight and more starch than the normal endosperm. Also, genetic manipulation of ADP-Glc PPase activity in potato tuber (70) and wheat endosperm (71) leads to increase of starch. This finding also has been shown for rice (72) as well as for cassava root (73). These results confirm that ADP-Glc synthesis is rate limiting for starch synthesis. Thus, data observed with the allosteric mutant ADP-Glc PPases of C. reinhardtii (64), maize endosperm (69), and Arabidopsis (66, 67) present strong evidence that in vitro allosteric effects are functional in vivo.

 

Plant ADP-Glc PPases can be activated by thioredoxin

ADP-Glc PPase from potato tuber has an intermolecular disulfide bridge that links the two small subunits by the Cys12 residue; it can be activated by reduction of the Cys12 disulfide linkage (46). At low concentrations (10 p,M) of 3-PGA, both spinach leaf reduced thioredoxin f and m reduce and activate the enzyme. Fifty percent activation was observed for 4.5- and 8.7- ^M reduced thioredoxin f and m (47). The activation was reversed by oxidized thioredoxin. Cys12 is conserved in the ADP-Glc PPases from plant leaves and other tissues except for the monocot endosperm enzymes. In photosynthetic tissues, this reduction may also be physiologically pertinent in the fine regulation of the ADP-Glc PPase.

Both potato tuber and potato leaf ADP-Glc PPases are plastidic; the leaf enzyme is in the chloroplast, and the tuber enzyme is in the amyloplast (74). The ferredoxin-thioredoxin system is located in the chloroplast and thus, with photosynthesis, reduced thioredoxin is formed and activated within the leaf ADP-Glc PPase. At night, oxidized thioredoxin is formed; it oxidizes and inactivates the ADP-Glc PPase. This activation/inactivation process during the light/dark cycle allows a fine tuning and dynamic regulation of starch synthesis in the chloroplasts. Thioredoxin isoforms are present in many different subcellular locations of plant tissues; cytosol, mitochondria, chloroplasts, and even nuclei (75) and are also present in amyloplasts (76).

It has been shown starch synthesis that occurs in potato tubers from growing plants is inhibited within 24 hours after detachment (77) despite having high in vitro ADP-Glc PPase activity and high levels of substrates, as well asATP and Glc-1-P and an increased 3-PGA/Pi ratio. In the detached tubers, the small subunit in nonreducing SDS-PAGE is solely in dimeric form and relatively inactive in contrast to the enzyme form of growing tubers where it was composed as a mixture of monomers and dimers. The detached tuber enzyme had a great decrease in affinity for the substrates as well as for the activator. Treatment of tuber slices with either DTT or sucrose reduced the dimerization of the ADP-Glc PPase small subunit and stimulated starch synthesis in vivo. These results indicate that reductive activation, which was observed in vitro of the tuber ADP-Glc PPase (46, 47), is important for regulating starch synthesis (77). A strong correlation between sucrose content in the tuber and the reduced/activated ADP-Glc PPase was noted.

 

Characterization of ADP-Glc PPases from some different sources

As previously indicated, Table 1 summarizes the kinetic and regulatory properties of purified potato tuber and spinach leaf ADP-Glc PPases. As reviewed in References 5 and 53, the properties of many other plant, algal, and cyanobacterial ADP-Glc PPases are similar. However, some ADP-Glc PPases within plant reserve tissue show some differences with respect to allosteric properties and their nonplastidic location. These examples are summarized below.

 

Barley

The barley leaf ADP-Glc PPase has been purified to homogeneity (69.3 U/mg), and it shows high sensitivity toward activation by 3-PGA and inhibition by phosphate (78). Substrate kinetics and product inhibition studies in the synthesis direction suggested a sequential Iso Ordered Bi Bi kinetic mechanism (78). ATP or ADP-Glc bind first to the enzyme in the synthesis or pyrophosphorolysis direction, respectively, which is similar to the E. coli enzyme (58).

Partially purified barley endosperm ADP-Glc PPase was shown to have low sensitivity to the regulators 3-PGA and Pi (78). However, 3-PGA decreased up to threefold the S0.5 for ATP and the Hill coefficient. At 0.1-mM ATP, the activation by 3-PGA was around fourfold (79), and phosphate 2.5 mM reversed the effect. A recombinant enzyme with a (His)6-tag from barley endosperm was expressed using the baculovirus insect cell system (80). It shows no sensitivity to regulation by 3-PGA and Pi. However, the enzyme was assayed at saturating concentration of substrates and only in the pyrophosphorolysis direction. For ADP-Glc PPases, the synthetic direction is more sensitive to activation. When the recombinant enzyme without the (His)6- tag is expressed in insect cells, the heterotetrameric form still was not activated by 3-PGA nor inhibited by Pi at saturating levels of substrates (81). Whether 3-PGA had any effect on the affinity for the substrates as shown in the enzyme purified from the endosperm was not reported. Of interest is that the small (catalytic) subunit; when expressed alone it is very responsive to the allosteric effectors (81). This finding suggests that the large subunit in barley endosperm desensitizes the small subunit to activation by 3-PGA, and inhibition by Pi is opposite of what is observed for large subunits of potato tuber (42, 45) and Arabidopsis (56)

In green algae and in leaf cells of higher plants, ADP-Glc PPase has been demonstrated to reside in the chloroplast (82). More recently, using plastids isolated from maize and barley endosperm (83-85), the existence of two ADP-Glc PPases, a plastidial form, and a major cytosolic form were found. Subsequently, cytosolic forms of ADP-glucose pyrophosphorylase have been found in wheat (86, 87) and rice (88). Because starch synthesis occurs in plastids, it was proposed that in cereal endosperms, synthesis of ADP-Glc in the cytosol requires the involvement of an ADP-Glc carrier in the amyloplast envelope (85). Subsequently, characterization of the ADP-Glc transporter has been reported for maize endosperm (89, 90), barley endosperm (91), and wheat endosperm (92).

 

Pea embryos

ADP-Glc PPase from developing embryos of peas was purified to apparent homogeneity (56.5 U/mg), and it was found to be activated up to 2.4-fold by 1-mM 3-PGA in the ADP-Glc synthesis direction (93). In pyrophosphorolysis, 1-mM Pi inhibited the enzyme 50%, and 3-PGA reversed this effect. The effect of 3-PGA or Pi on the S0.5 for ATP was not analyzed.

 

Maize endosperm

Partially purified maize endosperm ADP-Glc PPase (34 U/mg) was found to be activated by 3-PGA and Fru 6-P (25- and 17-fold, respectively) and inhibited by Pi (94). The heterotetrameric endosperm enzyme has been cloned and expressed in E. coli, and its regulatory properties were compared with an isolated allosteric mutant less sensitive to Pi inhibition (69). As indicated above, the increase of starch noted in the mutant maize endosperm ADP-Glc PPase insensitive to Pi inhibition supports the importance of the allosteric effects of 3-PGA and Pi in vivo. Also as indicated above, it is believed that the major endosperm ADP-Glc PPase isoform is located in the cytosol (83).

 

Identification of Important Amino Acid Residues Within the ADP-Glc PPases

Amino acid residues that play important roles in the binding of substrates and allosteric regulators have been identified in the ADP-Glc PPases mainly by chemical modification and site-directed mutagenesis studies. Thus, photoaffinity analogs of ATP and ADP-Glc, 8-azido-ATP, and 8-azido-ADP-Glc were used to identify Tyr114 as an important residue in the enzyme from E. coli (95, 96). Site-directed mutagenesis of this residue rendered a mutant enzyme that exhibits a marked increase in S0.5 for ATP as well as a lower apparent affinity for Glc-1-P and the activator Fru1,6-bisP (95). The Tyr residue must be close to the adenine ring of ATP or ADP-Glc but probably also near the Glc-1-P and the activator regulatory sites. The homologous Tyr114 in the enzyme from plants is a Phe residue (54), which suggests that the functionality is not given by the specific residue but by its hydrophobicity.

Chemical modification studies on the E. coli ADP-Glc PPase that showed involvement of Lys195 in the binding of Glc-1-P (97, 98) were confirmed by site-directed mutagenesis (99). Site-directed mutagenesis was also used to determine the role of this conserved residue in the small-subunit Lys198 and large-subunit Lys213 of the potato tuber ADP-Glc PPase (44). Mutation of Lys198 of the small subunit with Arg, Ala, or Glu decreased the apparent affinity for Glc-1-P 135-fold to 550-fold. Little effect is observed on kinetic constants for ATP, Mg2+, 3-PGA, and Pi. The results show that the Lys198 in the small subunit is involved directly in the binding of Glc-1-P. However, the homologous site residue Lys213 in the large subunit does not seem to be involved because similar mutations on Lys213 had little effect on the affinity for Glc-1-P (44). This finding is consistent with the view that the potato tuber large subunit is a modulatory subunit and does not have a catalytic role (42).

Asp142 in E. coli ADP-Glc PPase in modeling studies was predicted to be close to the substrate site, and this amino acid was identified as mainly involved in catalysis (60). Site-directed mutagenesis of D142 to D142A and D142N confirmed that the main role of Asp142 is catalytic for a decrease in specific activity of 10,000-fold was observed with no other kinetic parameters showing any significant changes (60). This residue is highly conserved throughout ADP-Glc PPases from different sources as well as throughout the super-family of nucleotide-sugar pyrophosphorylases (59). The role of this Asp residue was also investigated by site-directed mutagenesis in the heterotetrameric potato tuber ADP-Glc PPase. The homologous residues of the small-subunit Asp145 and large-subunit Asp160 were replaced separatley by either Asn or Glu residues (59). Mutation of the Asp145 of the small subunit rendered the enzymes almost completely inactive. D145N mutant activity decreased by four orders of magnitude, whereas D145E, which is a more conservative mutation, decreased in specific activity by just two orders of magnitude. The homologous mutations in the large subunit alone (D160) did not alter the specific activity, but they did affect the apparent affinity for 3-PGA (59). Thus, these results agree with the view that each subunit in potato tuber ADP-Glc PPase plays a particular role: the small subunit conducts catalysis and the large subunit plays a regulatory role.

Pyridoxal-5-phosphate (PLP) could be considered to have some structural analogy to 3-PGA, and it was found to activate both the enzymes from spinach leaf and Anabaena. In spinach ADP-Glc PPase, PLP bound at Lys440, which is very close to the C-terminal of the small subunit, and bound to three Lys residues in the large subunit. Binding to these sites was prevented by the allosteric effector 3-PGA, which indicated that they are close to or are involved directly in the binding of this activator (100, 101).

With Anabaena ADP-Glc PPase, PLP modified Lys419 and Lys382. That these residues were regulatory binding sites was confirmed by site-directed mutagenesis of the Anabaena ADP-Glc PPase (102, 103). Mutation of the homologous Lys residues in the potato tuber enzyme small-subunit Lys441 and Lys404 indicated that they were also part of the 3-PGA site in heterotetrameric ADP-Glc PPases and that they contribute additively to the binding of the activator (45). Moreover, mutation on the small subunit yielded enzymes with lesser affinity to 3-PGA, measured by 3090-fold and 54-fold, respectively, than the homologous mutants on the large subunit. Results indicate that Lys404 and Lys441 on the potato tuber small subunit are more important than their homologous counterparts on the large-subunit Lys417 and 455. It seems that the large subunit seems to contribute to the enzyme activation by making the activator sites already present in the small subunit more efficient rather than by providing more effective allosteric sites (45).

Arginine residues were found in ADP-Glc PPases from cyanobacteria to be functionally important as shown by chemical modification with phenylglyoxal (104, 105). Also, the role played by Arg294 in the inhibition by Pi of the enzyme from Anabaena PCC 7120 was shown by Ala-scanning mutagenesis studies (105). More recently, it was found that replacement of this residue with Ala or Gln produces mutant enzymes with a changed pattern of inhibitor specificity; it was found that they have NADPH rather than Pi as the main inhibitor (106). All these results suggest that the positive charge of Arg294may play a key role in determining inhibitor selectivity rather than being specifically involved in Pi binding. However, studies on the role of Arg residues located in the N-terminal of the enzyme from Agrobacterium tumefaciens demonstrated the presence of separate subsites for the activators Fru-6-P and pyruvate as well as a desensitization of R33A and R45A mutants to Pi inhibition (107).

Random mutagenesis experiments performed on the potato tuber ADP-Glc PPase have been useful to identify residues that are important for the enzyme. Mutation of Asp253 on the small subunit showed a specific effect on the K m for Glc-1-P, which increased 10-fold with respect to the wild-type enzyme (108). The small magnitude in the increase (only one, rather than three to four orders of magnitude) suggests that the Asp253 residue is not involved in directly Glc-1-P binding. This residue, however, is conserved in the NDP-sugar PPases that have been crystallized and the structure solved (61-63). Alignment of Asp253 in the structure according to the secondary structure prediction (109) places the residue close to the substrate site without direct interaction with Glc-1-P suggests that substitution of the Asp 253 causes an indirect effect on the Glc-1-P by alteration of the Glc-1-P-binding domain. Another random mutagenesis study (110) concerns Asp416 (described in the article as Asp413) of the potato tuber ADP-Glc PPase small subunit and its effect on 3-PGA activation. This residue is adjacent to Lys417, which has been shown to be a site for PLP binding and 3-PGA activation (100). Also, several modifications on the C-terminus caused modifications on the regulation of different plant ADP-Glc PPases (69, 111).

The finding that Lys and Arg residues are important in allosteric effector binding and are situated at the C-terminus in ADP-Glc PPases of plants and cyanobacteria is different with what is observed for the bacterial ADP-Glc PPases. Lys39 (E. coli) and Arg residues in the N-terminal of the A. tumefaciens enzyme were shown to be important for the interaction of the activators and inhibitors (97, 98, 107, 112). These results suggest that the regulatory domains may be at different sites in the bacterial and plant enzymes. Other studies, however, with chimeric ADP-Glc PPases constructed from E. coli and A. tumefaciens have shown that the C-terminus in the bacterial ADP-Glc PPases are also functional in determining effector specificity and affinity (113). Regulation of ADP-Glc PPases most likely is determined by interactions that occur between the N- and C-termini in the enzyme.

 

Characterization of the regulatory domain

Truncation of 10 amino acids in the small subunit of potato tuber ADP-Glc PPase affected its regulatory properties by increasing the apparent affinity for the activator 3-PGA and decreasing the inhibitor Pi affinity (42). When the large (regulatory) subunit was truncated by 17 amino acids at the N-terminal, similar results were observed (114). The regulatory properties of the E. coli enzyme were also affected when the N-terminal is shortened by 11 amino acids (115, 116). The N-terminal region of the ADP-Glc PPase is predicted to be a loop, and the data suggests it regulates enzyme activity by acting as the “allosteric switch”; it is involved in the transition between the activated and nonactivated conformations of the enzyme. A shorter N-terminus allows the enzyme to be in an “activated” conformation.

 

Starch Synthases and Branching Enzymes

Starch synthase catalyzes transfer of the glucosyl moiety of ADP-glucose either to a maltosaccharide or to the starch polymers, amylase, and amylopectin, which form a new α-1,4-glucosidic linkage (reaction 2). The glucosyl anomeric linkage in ADP-glucose is a, and thus the newly formed glucosidic linkage is retained. The starch synthase is therefore characterized as retaining GT-B glycosyltransferase (117). More than one form of starch synthase is present in many plant tissues. This finding has been summarized in several publications (1-4, 118,), and the starch synthases are encoded by more than just one gene. Some starch synthases are bound to the starch granule and are designated as starch granule-bound starch synthases. They may be solubilized by α-amylase digestion of the granule, whereas others designated as soluble starch synthases can be found in the soluble portion of the plastid fraction.

Starch synthase or glycogen synthase activity can be measured by transfer of [14C]glucose from ADP-glucose into an appropriate primer such as amylopectin or rabbit glycogen and then precipitation of the labeled polymer with ethanol (119).

 

Characterization of the starch synthases

A phylogenetic tree based on the various deduced amino acid sequences of the plant starch synthases and green alga, Chlamydomonas, have identified five subfamilies of starch synthases (2). These subfamilies are known as granule-bound starch synthase (GBSS), starch synthase 1 (SS1), starch synthase 2 (SS2), starch synthase 3 (SS3), and starch synthase 4 (SS4). Additional data indicates that the SS2 class may have diverged even more to classes SS2a and SS2b (120). Also, the GBSS family may have also diverged into another class, GBSS1, GBSS1b or GBSS2 (121-123). The starch synthases are reviewed in detail in Reference 124.

 

Branching enzyme

As with starch synthases, many isozymes have been found for the Branching Enzyme, and they have been characterized in a number of plants.

Maize endosperm has three branching enzyme (BE) isoforms (24, 25) BE I, Ila, and Ilb from maize kernels (3, 125, 126). Molecular weights were 82 KDa for BEI and 80 KDa for BEs Ila and IIb. Table 6 summarizes the in vitro properties of the various maize endosperm BE isozymes from the studies of Takeda et al. (127) and Guan et al. (24, 25). BEI had the highest activity in branching amylase, and its rate of branching amylopectin was about 3% observed with amylose. The BEIIa and BEIIb isozymes branched amylopectin at twice the rate they branched amylase, and they catalyzed branching of amylopectin at 2.5 to six times the rate observed with BEI. BEI catalyzes the transfer of longer branched chains, and BEIIa and IIb catalyze the transfer of shorter chains (127). This finding may suggest that BEI produces a slightly branched polysaccharide that serves as a substrate for enzyme complexes of starch synthases and BEII isoforms to synthesize amylopectin. BE II isoforms may also play a predominat role in forming the short chains present in amylopectin. Moreover, BEI may be more involved in producing the more interior B-chains of the amylopectin, whereas BEIIa and BEIIb would be involved in forming the exterior (A) chains.

Potato branching enzyme thus shows a high degree of similarity to maize BEI and to maize BEII isozymes, however to a lesser extent (128, 129). As observed with the maize-branching enzymes, potato BEI was more active on amylose than BEII, and BEII was more active on amylopectin than BEI (130-132).

Branching isoenzymes from rice (21, 133), wheat (134, 135), and barley (136, 137) have also been characterized. The effects on starch structure in plants that have mutations in the starch-branching enzymes have been reviewed (2-4, 118, 124).

 

Table 6. Properties of maize endosperm-branching isozymes

 

Branching enzymes

BEI

BEIIa

BEIIb

Phosphorylase stimulation (a)

1196

795

994

Branching linkage assay (b)

Iodine stain assay (c)

2.6

0.32

0.14

Amylose (c1)

800

29.5

39

Amylopectin (c2)

24

59

63

Ratio of activity a/b

460

2484

7100

a/c1

1.5

27

25

a/c2

49.8

13.5

15.8

c2/c1

0.03

2

1.6

Phosphorylase stimulation and branching linkage assays units are ^mol/min (126). The iodine stain assay unit is (127), a decrease of one Absorbance unit per min.

 

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Further Reading

Starch Structure

Bertoft E. Analyzing starch structure. In: Starch in Food: Structure, Function and Applications. Eliasson A-C, ed. Woodhead Publishing Limited, Boca Raton, FL. pp. 57-96.

French D. Organization of starch granules. In: Starch: Chemistry and Technology, 2nd edition. Whistler RL, BeMiller JN, Paschall EF, eds. Academic Press, Orlando, FL. pp. 183-247.

A review on Maize Muatants with Altered Starch Structure

Shannon, JC, and Garwood, DL. Genetics and physiology of starch development. In: Starch: Chemistry and Technology, 2nd edition. Whistler RL, BeMiller JN, Paschall EF, eds. Academic Press, Orlando, FL. pp. 25-86.

A book with many good reviews on source-sink relationships and factors affecting source-sink interactions

Zamski E, Schaffer AA, eds. Photoassimilate Distribution in Plant and Crops: Source-Sink Relationships. 1996.

Other Reviews on Starch Metabolism

Smith AM, Denyer K, Martin C. The synthesis of the starch granule. Ann. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48:67-87

Smith AM, Zeeman SC, Smith SM. Starch degradation. Ann. Rev. Plant Biol. 2005; 56:73-98.

Stitt M. Regulation of metabolism in transgenic plants. Ann. Rev. Plant Physiol. 1995; 46:341-368.