Critical Review A Review of Starch-branching Enzymes and Their Role in Amylopectin Biosynthesis

Ian J. Tetlow* Michael J. Emes

Department of Molecular and Cellular Biology, Science Complex, University of Guelph, Guelph, ON, Canada

Abstract Starch-branching enzymes (SBEs) are one of the four major enzyme classes involved in starch biosynthesis in plants and algae, and their activities play a crucial role in determining the structure and physical properties of starch granules. SBEs generate a-1,6-branch linkages in a-glucans through cleavage of internal a-1,4 bonds and transfer of the released reducing ends to C-6 hydroxyls. Starch biosynthesis in plants and algae requires multiple isoforms of SBEs and is distinct from glycogen biosynthesis in both prokaryotes and eukaryotes which uses a single branching enzyme (BE) isoform. One of the unique characteristics of starch structure is the grouping of a-1,6-branch points in clusters within amylopectin. This is a feature of SBEs and their interplay with other starch biosynthetic enzymes, thus facilitating formation of the compact water-insoluble semicrystalline starch granule. In this respect, the activity of SBE isoforms is pivotal in starch granule assembly. SBEs are structurally related to the aamylase superfamily of enzymes, sharing three domains of sec-

ondary structure with prokaryotic Bes: the central (b/a)8-barrel catalytic domain, an NH2-terminal domain involved in determining the size of a-glucan chain transferred, and the C-terminal domain responsible for catalytic capacity and substrate preference. In addition, SBEs have conserved plant-specific domains, including phosphorylation sites which are thought to be involved in regulating starch metabolism. SBEs form heteromeric protein complexes with other SBE isoforms as well as other enzymes involved in starch synthesis, and assembly of these protein complexes is regulated by protein phosphorylation. Phosphorylated SBEIIb is found in multienzyme complexes with isoforms of glucan-elongating starch synthases, and these protein complexes are implicated in amylopectin cluster formation. This review presents a comparative overview of plant SBEs and includes a review of their properties, structural and functional characteristics, and recent developments on their postC 2014 IUBMB Life, 66(8):546–558, 2014 translational regulation. V

Keywords: amylopectin; amylose; branching enzyme; glycoside hydrolase family; polyglucan; protein complexes; protein phosphorylation; starch-branching enzyme; starch; starch synthesis.

Abbreviations: ae, amylose extender; BE, branching enzyme (1,4-a-glucan: 1,4-a-glucan 6-glucosyl transferase); C-terminus, carboxyl terminus; CBM48, family 48 carbohydrate-binding module; CDPK, Ca21-dependent protein kinase; DBE, debranching enzyme; DP, degree of polymerization; GBE, glycogen-branching enzyme; GH13, glycoside hydrolase family 13; MOS, maltooligosaccharides; NH2-terminus, amino terminus; SBE, starchbranching enzyme; Ser, serine; SP, starch phosphorylase; SS, starch synthase. This work is dedicated to the memory of Koushik Seetharaman. C 2014 International Union of Biochemistry and Molecular Biology V Volume 66, Number 8, August 2014, Pages 546–558 *Address correspondence to: Ian J. Tetlow, Department of Molecular and Cellular Biology, Science Complex, University of Guelph, Guelph, Ontario, Canada N1G 2W1. Tel: 1 519 824 4120 extension 52735 Fax: 1 519 837 1802 E-mail: [email protected] Received 6 June 2014; Accepted 7 August 2014 DOI 10.1002/iub.1297 Published online 5 September 2014 in Wiley Online Library (wileyonlinelibrary.com)

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Introduction Starch produced in the plastids of higher plants represents the most abundant storage polyglucan in nature, functioning as a short- and long-term reserve carbohydrate. Starch is made up of linear a-1,4-linked glucans synthesized by starch synthases (SS, E.C. 2.4.1.21), a group of ADP-glucose-dependent transferases (1), and a-1,6-linked branches, which allow a high level of packing of glucan chains within the polymer and no adverse effects on the osmotic pressure of the cell. Starch-branching enzymes (SBEs; 1,4-a-glucan: 1,4-a-glucan 6-glucosyl transferase; E.C. 2.4.1.18) influence the structure of starch by catalyzing the formation of a-1,6-branch points with varied frequency and branch chain length. The activity of BE was first identified in potato by Haworth et al. (2). As phosphorylase (E.C. 2.4.1.1, or P-enzyme) was thought to be solely responsible for a-1,4glucan chain elongation in glycogen and amylopectin, the branching activity was termed Q-enzyme. The relative

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frequency of a-1,6-branch points, as well as their positioning relative to one another, has an important influence on the physicochemical properties of starch and glycogen and is largely a reflection of their respective biological functions. Starch granules consist of tightly packed glucan chains resulting in a semicrystalline, water-insoluble structure, which is ideally suited for long-term storage. The various SBE isoforms in plants impact structural and functional properties of starches. For example, in crop plants, the activities of different SBE isoforms govern a number of important end uses, for example in the food industry, where digestibility and processing characteristics can be controlled by branching frequency and branch chain length. Modification of SBEs in planta also influences the degradation of starch reserves in developing seeds, thus impacting seedling vigor (see later). This review provides a current overview of SBEs and their role in starch biosynthesis. Structure/function relationships of SBEs will be discussed in relation to starch granule formation, including how specific roles for different isoforms in the pathway of starch biosynthesis have been assigned through study of mutants. In this article, the current knowledge on the regulation of SBEs is outlined, including their ability to form protein complexes with other enzymes as well as their regulation by protein phosphorylation.

Mode of Action and Properties of SBEs BEs catalyze the transglycosylation of an a-1,4-glucosidic linkage in an 1,4-a-D-glucan to form a nonreducing-end maltooligosaccharide (MOS) chain, which is then transferred to a C-6 hydroxyl group in a nonreversible reaction (3) resulting in the formation of a-1,6-branch points within a-1,4-polyglucans. The cleaved glucan can be transferred to an acceptor chain, which is either part of the original glucan chain (intrachain transfer) or to part of an adjacent glucan chain (interchain transfer; see Fig. 1). It has been thought that the close association of glucan chains, in a double-helical configuration, creates a more favorable environment for the interchain transfer (4,5). The addition of new branch points by SBE produces new nonreducing ends for glucan-elongation reactions by SS or starch phosphorylase (SP), indicating that SBEs determine polymer structure and impact the amount of a-glucan synthesized through their influence on other enzyme activities. In addition, SS activity can be stimulated in the presence of SBEs, particularly when there is no added primer (see ref. 6, and references therein). SBEs are catalytically active as monomeric proteins. However, recent evidence in cereal endosperm amyloplasts indicates that they can associate with other starch biosynthetic enzymes (7,8) and can also form dimers (see “Regulation of SBE Activity” section; ref. 9. Plant SBE isoforms show wide variation in chain-length transfer pattern during catalysis related to their glucan substrate preferences (see Table 1), characteristics which are dictated by structural features of the amino- (NH2-) and carboxyl-

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(C-) termini, respectively (see “Structure–Function Relationships of SBEs” section). The different catalytic and kinetic characteristics associated with plant SBEs have implications for their different roles in starch synthesis. Minimum chainlength requirement for branching differs between the SBEI and SBEII classes (13,28). The minimum glucan chain-length requirement for branching by maize (Zea mays L.) SBEI is a degree of polymerization (DP) of 15, whereas for the SBEII class, it is DP 12. Given that the minimum DP requirement for linear MOS to form a double-helical configuration is DP 10, the double helix may be a requirement for catalytic action by plant SBEs. SBEI and SBEII classes also differ in their preference for the length of a-glucan chain transferred. The most extensive work on the characteristics of plant SBEs was with maize endosperm enzymes in the 1990s. These studies showed that SBEI has a preference for amylose as a substrate and transfers relatively longer glucan chains (up to DP 30, with the majority being DP 10–13; refs. (11) and (12)). In cereals, the SBEII class is subdivided into two distinct gene products, SBEIIa and SBEIIb, each with different kinetic characteristics and tissue expression patterns. SBEII isoforms transfer shorter chains (DP 6–14) and prefer amylopectin as a substrate (29). These characteristics have also been shown in a variety of other plant species, for example, rice (Oryza sativa L.; ref. (15), wheat (Triticum aestivum L.; ref. 20, and potato (30). SBEIIb in monocots is found exclusively in nonphotosynthetic storage (endosperm) tissues and has a narrower range of glucan transfer preference (DP 6–7) than SBEIIa, which is ubiquitously expressed; these features may be related to differences in the fine structure of starches from different sources, for example, leaf versus endosperm starch, or in tissues expressing varied proportions of the two isoforms (15). In vitro experiments with SBEs have shown that during extended incubation times, SBEs initially transfer longer MOS to acceptor chains, which are subsequently used as a donor substrate, producing short-branch side chains (12). The interplay between the various isoforms of SBE and SS and the a-glucan structures they form has implications for our understanding of starch biosynthesis in plants and algae (see later).

SBEs Play a Crucial Role in Starch Biosynthesis Higher plants and algae synthesize starch through the coordinated actions of multiple isoforms of SBE, and the structures formed by SBEs are modified by debranching enzymes (DBEs) to form crystalline-competent, water-insoluble structures. By contrast, glycogen synthesis depends on a single BE isoform to introduce a-1,6-branch points within the particle. Multiple SBEs activities are therefore critical determinants of the branching pattern in amylopectin and the polymodal distribution of chain lengths underpinning its cluster structure (31,32). Gene expression studies with a range of species indicate that the SBEI class is expressed later in development than the SBEII class (see ref.

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FIG 1

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Scheme of the two reaction mechanisms of SBE on linear a-1,4-linked glucans showing Haworth projection of glucose moieties. A: Interchain branching: Cleavage of a a-1,4-linkage in a donor chain and transfer of the nonreducing end-terminal fragment to the C-6 hydroxyl position of a glucosyl residue on an adjacent acceptor chain to create a a-1,6-glucosidic (branch) linkage. Interchain branching reaction probably predominates in plant SBEs. B: Intrachain transfer: Cleavage of an a-1,4glucosidic linkage followed by transfer of the nonreducing end-terminal fragment to the C-6 hydroxyl position of a glucosyl residue on the same chain. Different SBE isoforms have distinct preferences for chain-length transfer. The glucosyl residue used as an acceptor for the synthesis of the a-1,6-glucosidic (branch) linkage is shaded in gray; (!) site of the a-1,4-glucosidic linkage cleaved by SBE. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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86

SBE 2.2

90 85

SBEIIa

SBEIIb

82

85

SBEI (RBEI)

SBEIIa (RBE4)

Rice (Oryza sativa)

86

SBEI

Maize (Zea mays)

93.5

SBE 2.1

Theoretical mass (kDa)

i

d

d

1.2 (AS1160), 31 (AS55),

0.18 (AS1160), 2.9 (AS55), 18 mg/ml (ae2amylopectin)

11 (amylose DP405), 50 (amylose DP197)

10 (amylose DP405), 50 (amylose DP197)

2 (amylose DP405), 4.1 (amylose DP197), 50 (amylose DP56)

N.D.

N.D.

Km (lM)

Properties and characteristics of plant starch-branching enzymes

Arabidopsis (Arabidopsis thaliana)

Plant SBEs

TABLE 1

927, b0.33

a

a

2700 (AS1160) 1200 (AS55),

3000 (AS1160), 2300 (AS55), 2000 (ae2 amylopectin)

795, b0.32

a

a

1332, b2.4

a

N.D.

N.D.

Vmax (Units mg21)

6 to 15

6 to 15 or longer (6 and 11)

3-9 (DP 6-7)

DP 30 (DP10-13)

N.D.

N.D.

Chain length (DP) transfer range (preference)

Enhances activity of plastidial SP

Phosphorylated at Ser286, Ser297 and Ser649; forms protein complexes with soluble starch synthases and other SBEs

Phosphorylated on Ser residue(s), forms protein complex with SBEIIb and starch phosphorylase

activity redox modulated

activity redox modulated

Regulatory characteristics

(15)

(15–19)

(7,9,11,14)

(7,12,13)

(11–13)

(10)

(10)

Reference

550

BEI

Kidney bean (Phaseolus vulgaris)

SBEI

64-85

80

f

1.27 mg ml21 (potato amylose)

N.D

N.D

SBEIIb

Potato (Solanum tuberosum)

N.D

SBEIIa

0.65 mg ml21

0.11 mg ml21 (WBE IB)

0.3 mg ml21 (WBE IAD)

0.78 (AS1160), 2.6 (AS55), 1.3 mg/ml (ae2amylopectin)

N.D

e

e

e

d

1.5 mg/ml (ae2amylopectin)

Km (lM)

SBEI

85

88

SBEII

Barley (Hordeum vulgare)

87

SBEI

Wheat (Triticum aestivum)

82

SBEIIb (RBE3)

i

Theoretical mass (kDa)

(Continued)

Plant SBEs

TABLE 1

186

121

322

190 (AS1160) 45 (AS55), 500 (ae2 amylopectin)

c

c

251

4.62

N.D

N.D

N.D

a

a

a

a

2

5400 (ae amylopectin)

Vmax (Units mg21)

12 to 18

10 to 12

N.D.

13-20 (DP16)

h

h

6 to 7

Chain length (DP) transfer range (preference)

Interaction with 14-3-3 protein

Interaction with 14-3-3 protein

Phosphorylated at serine(s); activity enhanced by phosphorylation

Phosphorylated at serine(s)

Regulatory characteristics

(26)

(25)

(18,21–24)

(21,22)

(7,20)

(7,20)

(15)

Reference

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21 c

c

c

c

c

135

561

396

242

234

Vmax (Units mg21)

N.D.

N.D.

5-40 (6)

5-40 (6)

N.D.

Chain length (DP) transfer range (preference)

Regulatory characteristics

(27)

(27)

(26,27)

(27)

(26,27)

Reference

N.D., not determined. a Measured by the phosphorylase a-stimulation assay. b Measured by determination of reducing ends (branch-linkage assay). c Measured by the iodine-staining assay. d Kinetic parameters of rice SBEs determined and compared using synthetic amyloses AS-1160 (DP 6,510) and AS-55 (DP 317) as described by Nakamura et al. (15). e Wheat SBE kinetics determined using amylose as substrate. f Variable masses likely a result of proteolysis during preparation. g Recombinant protein. h Chain-length transfer range deduced from starch analysis of mutants lacking respective SBE isoform. *Plant SBE masses are for mature (processed) form with transit peptide cleaved.

4.4 mgml21

4.8 mg ml

grLF-PvSBE2 (with amylopectin)

100

21

1.27 mg ml21

18.4 mg ml

rLF-PvSBE2 (with amylose)

93.8

0.74 mg ml21

Km (lM)

rPvSBE2 (with amylopectin)

g

g

rPvSBE2 (with amylose)

82

BEII (Pv SBE2)

g

i Theoretical mass (kDa)

(Continued)

Plant SBEs

TABLE 1

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FIG 2

Phylogenetic tree showing the amino acid sequence relationships of plant SBEs. Data are generated using BLAST-EXPLORER. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

33, and references therein). Phylogenetic analysis indicates that the SBEI gene probably evolved prior to the divergence of monocots and dicots (see Fig. 2; ref. 34. As noted above, the SBEIIa and b genes are differentially expressed; however, the endosperms of different cereals show considerable variation in the relative proportions of SBEIIa versus SBEIIb activity (16,35). In developing wheat endosperm, SBEIIb is expressed at much lower levels than SBEIIa (36), whereas in maize endosperm, SBEIIb predominates, being expressed at 50 times the level of the IIa form (35). SBEIIb is the most abundant protein in maize endosperm amyloplasts, whereas in leaf chloroplasts, the major BE isoform is SBEIIa (37,38). The analysis of mutant plants deficient in BE activity has shed much light on the roles of the various isoforms of SBE in amylopectin synthesis. It is noteworthy that loss of SBEII activity in many species often produces the clearest phenotypes; loss of SBEII activity causes the wrinkled (rugosus, r) phenotype in the pea (Pisum sativum L.) mutants studied by Mendel (39). The wrinkled phenotype of the seed in the r mutant is caused by an inhibition of starch biosynthesis (reduced by 50% when compared with wild-type seed) and a consequent accumulation of sucrose and other soluble sugars as the embryo develops, causing osmotic potential to rise, eventually causing wrinkling of the seed as it dries. In maize, loss of SBEIIb, the major isoform in the endosperm, produces the well-known amylose extender (ae2) mutation, resulting in a 20% reduction in starch synthesis and severely altered starch granule morphology; both ae2 and r starch granules are deeply fissured and irregularly shaped (40–42). The ae2 mutation in maize and rice produces a high-amylose starch, which is characterized by long internal chain lengths of amylopectin, and less frequently branched outer chains when compared with normal starches (ref. 33, and references therein). High-amylose starches are more resistant to a-amylase (E.C. 3.2.1.1) diges-

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tion than normal starches, leading to reduced digestibility (33), and are consequently regarded as high-value starches in the food industry. Production of high-amylose wheat starch involves suppression of both genes encoding SBEIIa and SBEIIb, resulting in starches containing >70% amylose, as suppression of SBEIIb alone caused no alteration in amylose content (43), and in potato, a high-amylose starch was produced by downregulation of both SBE isoforms (44). Interestingly, loss of SBEI activity in monocots and dicots has minimal apparent effects on starch synthesis and composition (45), although in a study by Yao et al., loss of SBEI from a SBEIIbdeficient background in maize was shown to cause increased branching of amylopectin. Yao et al. (46) postulated that this result was suggestive of a regulatory role for SBEI in influencing other SBE isoforms. Indeed, physical interactions between SBEI and SBEIIb have been reported in cereal endosperm amyloplasts (see later section). Maize SBEIIa mutants display a clear phenotype in leaf starch, but no measurable alteration in storage starch (37), suggesting a more important role for SBEIIa in transient starch synthesis in leaves but no critical role in endosperm amylopectin biosynthesis or one that is compensated by other SBEs. Studies with SBEIIa mutants of maize suggest that the isoform plays an important role in influencing starch structure to facilitate nocturnal degradation. Leaves of mutant plants were prematurely senescent, and the sparsely branched leaf starch produced in chloroplasts was ineffectively degraded over a diel cycle (38). Arabidopsis (Arabidopsis thaliana L.) has two SBEII-class enzymes termed SBE 2.1 and SBE 2.2 (47), plus a third unrelated form which is not expressed and has no known function. Deletion of either active isoform has minimal effect on starch synthesis, whereas simultaneous loss of both SBEII forms from Arabidopsis results in a failure to synthesize starch and accumulation of maltose in the cytosol (48). Some enzymes of starch biosynthesis are found strongly associated with starch granules, including SBEII isoforms from a range of species (49–52). The precise mechanism underlying the association of specific enzymes of starch synthesis with the starch granule are not known, although it has been suggested that alternative splicing of SBEII in bean (Phaseolus vulgaris L.) leads to partitioning within the starch granule (27). Recent evidence suggests that granule-associated proteins (including the SBEII class) become entrapped in the granule through association in heteromeric protein complexes (8,53). Distinct roles for SBEI and SBEII classes in amylopectin synthesis were suggested by the analysis of glucans produced when different combinations of maize SSs and SBEs were heterologously expressed in E. coli (54). The loss of SBEI in a number of studies in many species has failed to reveal a clear phenotype on starch structure and its accumulation, leaving its role in starch synthesis a somewhat open question. However, the analysis of germination of a SBEI-deficient mutant of maize revealed impaired seedling growth associated with inefficient a-amylase digestion of the starch reserves in the seed, highlighting the importance of starch granule architecture for physiological

Starch-Branching Enzymes In Amylopectin Biosynthesis

functions (55). The selection of SBEI in higher plants therefore appears to impart a survival advantage associated with seedling vigor and fitness. Starch is less branched than glycogen and has a polymodal glucan chain distribution with a hierarchical structure (31,32) arising from the coordinated action of a range of other biosynthetic enzymes including SSs and DBEs, all of which have multiple isoforms (33). Different SBE isoforms have been shown to strongly influence glucan structure. For example, transformation of a glycogen-branching enzyme (GBE)-deficient (glgBdeficient) E. coli with maize SBEs showed that the resulting glycogen structure was dependent on the particular SBE isoform complimenting the glgB mutant (28), supporting a role for different SBE isoforms in determining amylopectin fine structure. When GBE is incubated with amylose, the resulting glucan products resemble glycogen with respect to chain distribution (13), compatible with the irregular branching model of Hizukuri (31). Overexpression of SBEIIb in an SBEIIb-deficient mutant of rice produced a disorganized, highly branched hydrosoluble polyglucan (56), highlighting the importance of coordination and balance between SBE and SS activities in the plastid (see “Regulation of SBEs by Protein Phosphorylation” section). Amylopectin defines the structure of the starch granule, and granule architecture is remarkably conserved in the plant kingdom (32). In particular, amylopectin consists of clusters of linear a-1,4-glucans of DP 12–18, forming a crystalline lamella of variable size (4–6 nm) and an amorphous component consisting of clustered a-1,6-branch points. The cluster has an invariant size of 9 nm (57), and its crystalline glucan chains correspond to the minimum chain length required for branching by SBEI and SBEII (12), which suggests that SBEs may be critical in determining the amylopectin cluster size limit. It is thought that during amylopectin cluster formation, SBEII synthesizes new root clusters in concert with SS isoforms, that is, following elongation by SSIII, or the formation of long outer chains by SBEI to the products of SSIII (15). Following the actions of SSs and SBEs, DBEs are thought to be involved in facilitating packing of glucans to promote a water-insoluble product by removal of specific branch points (ref. 33, and references therein).

Structure–Function Relationships of SBEs Analysis of SBE primary sequences indicates that SBEs belong to the a-amylase superfamily of enzymes (termed the glycoside hydrolase family 13 [GH13]) and structurally made up of three domains: a central (b/a)-barrel catalytic domain, or A-domain, an NH2-terminal domain, and a C-terminal domain (58,59). The central catalytic domain is highly conserved among the members of the a-amylase (GH13) superfamily, and only isoamylases share all three domains with the BEs and are able to bind sugars in the a-1,6-position (59). The sequences of the NH2- and C-terminal domains are highly variable, but nevertheless jointly contribute to catalytic activity with the A-

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domain. Various experimental approaches (discussed in the sections below) have been used to deduce the functions of the three domains of SBE, including analysis of amino acid sequence alignments, site-directed mutagenesis (60), domainswapping experiments (12), and utilizing available X-ray crystallographic structures (59,61,62).

The Central Catalytic A-Domain The catalytic A-domain of SBEs is characterized by a symmetrical fold of eight parallel b-strands encircled by eight ahelices, which folds as a (b/a)8-barrel. X-ray crystallographic structures of GBE and SBE allowed elucidation of the (b/a)8barrel structural domain (59,61) and showed that variations occur in SBEs where the a-helix 5 is missing and replaced by extra a-helices (59). Another conserved feature of the central catalytic A-domain of SBEs is a group of four conserved amino acid regions (termed 1 to 4, see Fig. 3) also present on other GH13 enzymes. Site-directed mutagenesis experiments in a number of different organisms have been key to determining the importance of specific residues within each of the four domains. Seven highly conserved amino acids found in GH13 enzymes have been shown to be critical for catalysis; these are Asp376, His381, Arg445, Asp447, Glu502, His569, and Asp570 (maize SBEIIb numbering) and are shown in Fig. 3 in relation to their respective subdomains in the central region of these enzymes. Within this group of amino acids is a catalytic triad (Asp447, Glu502, and Asp570, located on regions 2, 3, and 4, respectively, of the catalytic A-domain, as shown in Fig. 3), whose significance was determined in site-directed mutagenesis studies with a recombinant maize SBEII enzyme (63,64). Mutation analysis and chemical modification studies with maize SBEIIb demonstrated the importance of the conserved Arg445 for catalysis (60) and showed that two conserved histidine residues (His381 in region 1 and His569 of region 4; see Fig. 3) were critical for substrate binding (65). Structural analysis of plant SBEs indicate that, in common with BEs from other organisms, catalytic capacity is shared between the Adomain, NH2-terminus, and C-terminus (see “The NH2-Terminal Domain” and “The C-Terminal Domain” sections).

The NH2-Terminal Domain SBEs and other GH13 enzymes, which cleave or form endo-a1,6-linkages, for example, isoamylase, pullulanase, share some structural similarities in the NH2-terminal domain, including a common, family 48, carbohydrate-binding module (CBM48; see Fig. 3; ref. 66). The biological function of the NH2-domain of BEs has been assigned through truncation and domain-swapping experiments with recombinant maize and E. coli enzymes. Preiss and coworkers conducted a number of experiments with chimeric and truncated maize SBEs to investigate the mechanisms underpinning the varied biochemical properties of plant SBE isoforms, including different chain-length transfer

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FIG 3

Functional domain organization of SBEs showing the amino- (NH2-) terminal domain (gray), the catalytic A-domain (black), and the carboxy- (C-) terminus (white). All BEs possess a family 48 carbohydrate-binding module (CBM48) within the NH2-terminus. The A-domain contains four highly conserved regions (1–4) and is a characteristic feature of the GH13 family of enzymes. The primary structures of the four regions from selected plant species is compared in the table below the domain organization diagram. The positions of key amino acid residues are given for each species; invariant amino acid residues are given in bold. Putative functional amino acid residues within regions were assigned by MacGregor et al. (58) and are given as follows: (• ) catalytic nucleophile; (䊏) acid/base catalyst; and (‡) transition-state stabilizer.

patterns (see above and Table 1). Chimeric maize enzymes were produced from SBEI, which transfers long glucan chains and a preference for amylose, and SBEIIb, which transfers short- to medium-length a-glucan chains, to determine which regions of the enzyme are responsible for chain-length recognition and branching. Production of one such chimeric enzyme (termed mBE I-II), consisting of the NH2-terminus of SBEI and the central (A-domain) and C-terminus of SBEIIb, resulted in an enzyme of low catalytic activity with chain transfer properties similar to SBEI, that is, producing branches of DP 11–12. As only the NH2 region of SBEI protein was present in mBE III, the data support a role for the N-terminus in determining the chain length of MOS transferred during catalysis.

The C-Terminal Domain Similar truncation experiments with maize SBEs were used to determine the importance of the C-terminus in the BE reaction. A chimeric SBE from maize enzymes was produced, replacing the C-terminal 229 amino acids of SBEIIb with the corresponding 284 amino acids of SBEI; the resulting protein was termed mBE II-I (12). The chimeric mBE II-I protein showed higher (threefold) catalytic activity than the wild-type enzyme and, like the SBEI protein, a preference for amylose. Analysis of the glucan products of the mBE II-I reaction showed an increase in the proportion of short (DP 6) chains transferred. As SBEIIb tends to transfer chains of around DP 6, the data support the suggestion that SBE’s C-terminus determines both substrate preference and catalytic activity. Further experiments involved truncation of 58 amino acids from the C-terminus of mBE II-I (67), producing an enzyme with similar Vmax for branching activity and Km for reduced amylose AS320. The truncated mBE II-I retained its preference

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for amylose over amylopectin, implying that the C-terminal SBEI extension is not required for catalysis or substrate preference. Although a further 87-amino acid deletion from the Cterminus of the truncated mBE II-I lost its catalytic activity, replacing the 87 amino acids of SBEI with the corresponding 79 amino acids from SBEIIb partially restored activity (25% of mBE II-I activity) and had no impact on substrate preference. These results suggest that the C-terminal region is required for catalytic activity (Leu649 to Asp735 of mBE II-I) and that another C-terminal domain (Gln510 to Asp648 of mBE II-I, corresponding to Gln476 to Asp614 of SBEI) is involved in determining substrate preference (67). Overall, the various studies with chimeric SBEs and truncation experiments point to a dual role for the C-terminus in controlling enzyme catalysis and in determining glucan substrate preference and the N-terminus governing glucan chain length transferred. It is also clear that some of these functions are modulated by other regions of the protein and that the conserved central (b/a)8-barrel domain is not solely responsible for catalytic activity.

Regulation of SBE Activity Coordinate expression of SBE genes is a feature of all species studied to date and was dealt with earlier. This section will focus on recent evidence which indicates that SBEs are subject to regulation at the post-translational level. A study by Morell et al. (20) revealed that SBE activity can be modified by certain solutes. In particular, physiological concentrations of specific anions, inorganic phosphate, and phosphorylated metabolites were able to stimulate partially purified SBEI and SBEII from wheat endosperm, whereas neutral sugars caused no change in activity. The general activation caused by phosphorylated

Starch-Branching Enzymes In Amylopectin Biosynthesis

FIG 4

Phosphorylation sites on maize SBEIIb and SBEIIa. A: Ser286 and Ser297 show high sequence conservation within the branching enzymes of the plant kingdom (14), whereas Ser649 is found only in the endosperm-specific SBEIIb isoform of some cereals (B). Molecular simulation studies indicate that phosphorylation of Ser297 forms a salt bridge with Arg665. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

compounds was thought to be due to stabilization of the enzymes’ active sites. Citrate can activate SS activity, by lowering the Km for the a-glucan primer, and can stimulate both spinach leaf (Spinacia oleracea L.) and bean (Phaseolus vulgaris) SBEs (6,26). The activity of a number of starch biosynthetic enzymes has been shown to be modified by redox state, and activities of Arabidopsis SBE 2.1 and 2.2 were enhanced under reducing conditions (10). Redox modulation of the pathway may play a role in diurnal starch regulation in leaves, although its role in regulation of storage starch biosynthesis in nonphotosynthetic tissues is not clear.

Regulation of SBEs by Protein Phosphorylation There is growing evidence that SBE isoforms are regulated by protein phosphorylation. In wheat endosperm amyloplasts and leaf chloroplasts, both isoforms of SBEII are catalytically activated by phosphorylation at one or more serine (Ser) residues. Furthermore, in vitro dephosphorylation reduced the activity of SBEIIa and SBEIIb in amyloplasts and SBEIIa in chloroplasts, but had no measurable effect on the activity of SBEI (7). Studies with wheat and maize amyloplasts have shown that all SBE isoforms can be phosphorylated (8,9) and that protein phosphorylation regulates the association of SBE and SS isoforms in multienzyme protein complexes (see below; refs. 9,68,69). In terms of understanding the role played by protein phosphorylation in regulating the starch biosynthetic pathway, maize endosperm represents the best characterized system to date. Recent studies with maize SBEIIb, the major isoform of SBEII in the starch-storing endosperm, have shown that the enzyme can be phosphorylated at three Ser sites by two plastidial Ca21-dependent protein kinase (CDPK) activities (14,70). Two of the phosphorylation sites (Ser286 and Ser297) are highly conserved among the SBEII class (see Fig. 4) and located on opposite ends of the central catalytic b-barrel, ideally posi-

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tioned to interact with glucan substrates. Ser297 is conserved among all SBEs, suggesting a general regulatory role, and structural analysis of the consequences of phosphorylation at the three sites indicated that phosphorylated Ser297 forms a stable salt bridge with Arg665, part of a conserved cysteinecontaining domain found in all SBEs (see Fig. 4). The third phosphorylation site, Ser649, may have a more specialized function as this site appears only in some members of the cereal SBEIIb class (Fig. 4). Interestingly, in other plant SBEIIs, Ser649 is, in some cases, substituted by Asp or another phospho-Ser mimic. The regions flanking the 11-amino acid sequence containing Ser649 are highly conserved in all classes of SBE. It is notable that both the Ser649 and Ser286 motifs are completely absent from SBEI, again suggesting a distinct class of functions for these sites. There is evidence of competition for the different phosphorylation sites by the plastidial CDPKs, as an allelic mutant of amylose extender maize expressing a truncated form of SBEIIb lacking Ser286 and Ser297 becomes hyperphosphorylated at the remaining Ser649 residue (53). Phosphorylation of SBEs plays a critical role in the formation of heteromeric protein complexes with SSs and other SBE isoforms (see below). Preliminary evidence suggests that phosphorylation of Ser649 is not involved in the formation of heteromeric protein complexes with SSs, but instead may maintain SBEIIb in a free form in the stroma or in association with SBEI (Fig. 5; unpublished data).

SBEs Form Heteromeric Protein Complexes in Amyloplasts An emerging aspect of our understanding of the regulation of storage starch biosynthesis is the physical association of different classes of amylopectin-synthesizing enzymes in protein complexes within starch-synthesizing plastids and the important regulatory role played by protein phosphorylation (7,9). The SBEII class forms a functional trimeric protein complex with SSI and SSIIa, and this complex is implicated in

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FIG 5

SBEs in cereals are regulated by protein phosphorylation and form functional heteromeric protein complexes, some of which are involved in amylopectin cluster synthesis of storage starches. The figure summarizes the current knowledge on the role played by protein phosphorylation in the assembly of protein complexes involving SBE isoforms during storage starch synthesis and is based on the experimental data from maize endosperm. SBEIIb is the major isoform of SBEII in this tissue and is phosphorylated at three Ser sites by at least two Ca21-dependent protein kinases (CDPKs) in amyloplasts (14). Phosphorylation of SBEIIb by CDPK catalyzes the assembly of a trimeric protein complex between SSI, SSIIa, and SBEIIb. All three enzymes in this complex are catalytically active and thought to be involved in the synthesis of amylopectin clusters (shown as cylinders in the diagram) at the periphery of the nascent starch granule. The trimeric protein complex eventually becomes entrapped within the growing starch granule through the carbohydrate-binding domain of SSIIa. Disassembly of the various protein complexes occurs through dephosphorylation of the respective enzymes via uncharacterized plastidial protein phosphatases (PPase). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

amylopectin cluster biosynthesis, given the substrate preferences of the individual enzymes and the fact that all three enzymes in the isolated complex remain catalytically active (17). Evidence from developing wheat endosperm suggests that SBEII isoforms in the trimeric protein complex have greater affinity for amylopectin, the presumptive substrate (9). The components of the trimeric SSI/SSIIa/SBEIIb protein complex eventually become entrapped within the starch granule through the glucan-binding capacity of SSIIa (17). SBEIIb has also been detected in a protein complex with SBEI and SP (E.C. 2.4.1.1); however, there is no evidence that this complex becomes associated with the starch granule unless there is a mutation in SBEIIb (7,8,23). Assembly and disassembly of a number of heteromeric protein complexes involved in amylopectin biosynthesis are regulated by protein phosphorylation (see above; refs. (8) and (9)). In the soluble trimeric protein complex, found in cereal endosperm amyloplasts, SBEIIb is phosphorylated as is granule-associated SBEIIb, consistent with the observation that enzymes found in the trimeric protein complex become entrapped in the starch granule. Uncom-

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plexed SBEIIb is also found phosphorylated in the plastid stroma (8,53). Regulation of the starch pathway by protein phosphorylation and protein–protein interactions is axiomatic of a highly regulated metabolic process, ultimately impacting the plant’s carbon resource. The precise phosphorylation sites of SBEIIb involved in protein complex assembly are not yet known, but phosphorylation will alter the surface charge of the protein, critical for modulating protein–protein interactions. The formation of stabilizing salt bridges between phosphate groups such as at the Ser297 site, and basic residues within disordered loop regions, could play an important role in constraining these loops, possibly orientating them for proteinbinding interactions.

Conclusions and Future Directions The ability to store carbon in the form of osmotically inert polyglucans is crucial to the survival of many organisms in nature and is only possible through the actions of BEs. The formation of starch granules in plants requires the careful coordination

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and control of many isoforms of SS, SBE, and DBE to form clusters of amylopectin, which are the building blocks of this structurally complex water-insoluble polymer. Our emerging understanding of structure/function relationships of SBEs has proved valuable in our appreciation of the roles of different SBE isoforms in the process of polyglucan biosynthesis and also provides an important foundation for understanding the effects of post-translational regulation of this enzyme class. SBEs are important targets for the improvement of starchstoring crops, in particular, as tools for the improvement of starch quality and functionality, for example, delivering health-beneficial high-amylose starches for the food industry. The recent discovery that SBEs are regulated by protein phosphorylation and form heteromeric protein complexes with other starch biosynthetic enzymes is an important development in our understanding of this important pathway. The identification of regulatory enzymes, plastidial protein kinases, and phosphatases involved in controlling protein–protein interactions will also be an important focus for future studies and provide valuable insight into the regulation of starch synthesis.

Acknowledgements The authors thank Dr. Fushan Liu for preparation of Figure 2 and the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) and Natural Sciences and Engineering Research Council (NSERC) for funding.

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