20.6: Biosynthesis of Starch, Sucrose and Cellulose

Search Fundamentals of Biochemistry

Learning Goals 

 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Sucrose Synthesis and Regulation

• Describe the cytosolic pathway for sucrose synthesis — tracing the conversion of triose phosphates to glucose-1-phosphate, then to UDP-glucose, and finally to sucrose-6-phosphate (catalyzed by sucrose-6-phosphate synthase using fructose-6-phosphate as acceptor) followed by phosphatase-mediated hydrolysis to sucrose — and explain why the acetal linkage between the anomeric carbons of glucose and fructose in sucrose makes it chemically stable (non-reducing, resistant to amylases, and unable to glycate proteins).
• Explain the regulatory logic by which fructose-2,6-bisphosphate (F2,6BP) coordinates sucrose synthesis with photosynthetic rate — describing how elevated DHAP and 3PG during active photosynthesis inhibit PFK2 to lower F2,6BP, thereby activating cytosolic FBPase-1 over PP-PFK-1 to increase fructose-6-phosphate availability for sucrose synthesis — and explain the opposite regulatory outcome in the dark.

Glycosyltransferase Mechanisms

• Describe the general mechanism of Leloir glycosyltransferases — including the ordered sequential bi-bi kinetic mechanism, the roles of Mn²⁺ in organizing the active site, and the stereochemical outcomes (inversion via S_N2 backside attack vs. retention via orthogonal attack through an oxycarbenium-like transition state) — and explain the structural basis for the different Mn²⁺ coordination geometries that distinguish inverting from retaining glycosyltransferases.
• Compare the three major glycosyltransferase fold families (GT-A, GT-B, GT-C) and explain how sucrose phosphate synthase and starch synthase, both GT-B enzymes, achieve their respective reactions through oxocarbenium ion-like transition states stabilized by conserved active-site residues.

Starch and Cellulose Biosynthesis

• Describe starch synthesis in plastids — contrasting it with glycogen synthesis (ADP-glucose vs. UDP-glucose as donor; addition to non-reducing vs. reducing end) — explaining the roles of ADP-glucose pyrophosphorylase (activated by 3PG and inhibited by Pi as the key regulated step), starch synthase (with processive vs. distributive mechanisms), branching enzyme, and debranching enzyme, and how redox regulation through thioredoxin-mediated disulfide bond cleavage activates key enzymes in the light.
• Describe the synthesis of cellulose by cellulose synthase (CesA) — explaining how 18 CesA trimers assemble into the large rosette-shaped cellulose synthase complex (CSC), how each trimer synthesizes one β(1,4)-linked glucan chain that is threaded through a transmembrane channel lined with aromatic and hydrophilic residues, and how the equatorial arrangement of all hydroxyl groups in cellulose enables extensive intermolecular hydrogen bonding to form rigid cellulose protofibrils.

Now that we have seen how carbon is captured and fixed into 3C trioses, which can be converted to fructose, glucose, and their derivatives, we can explore the synthesis of the key plant carbohydrates we all know: sucrose, starch, and cellulose.

The organization and some of the text derive from Sindayigaya and Longhini. https://www.peoi.org/Courses/Courses...chem/biochem18. Creative Commons - https://creativecommons.org/licenses...sa/3.0/deed.en

Sucrose Synthesis

Sucrose is a disaccharide of glucose and fructose with an acetal link between the anomeric carbons to form the nonreducing sugar O-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside. Its structure is shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Sucrose, a disaccharide of glucose and fructose

Sucrose can be considered a transport form of carbon, much like ketone bodies, which are transport forms of fatty acids. As noted above, the link between the sugars is between the anomeric C-1 link of glucose and the anomeric C-2 link of fructose. As such, it is not cleaved by typical carbohydrate-cleaving enzymes, such as amylases. Additionally, it doesn't react with proteins like other sugars with free cyclic hemiacetals, which can open and form reactive aldehydes. For example, the cyclic monosaccharide glucose, with its anomeric carbon in a readily reversible hemiacetal link, can form covalent bonds to amine groups in proteins such as hemoglobin, which forms the glycosylated version of HbA1c, as shown in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Reactions of cyclic hemiacetal sugars with amines. https://en.wikipedia.org/wiki/Glycated_hemoglobin. Creative Commons Attribution-ShareAlike License 3.0

The reaction proceeds via the formation of a Schiff base, followed by a rearrangement. HbA1c is a marker of diabetes. These features of sucrose may explain its selection as a key synthesized carbohydrate in plants.

Sucrose is synthesized in the cytosol, as shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Synthesis of sucrose in plants.

We have seen the enzymes that catalyze these reactions by forming G1P before in the glycolytic and gluconeogenic pathways. Glucose-1-phosphate is then converted to UDP-glucose, which reacts with fructose-6-phosphate to form sucrose 6-phosphate and UDP, catalyzed by the enzyme sucrose 6-phosphate synthase. The last reaction is the hydrolysis of sucrose 6-phosphate by sucrose 6-phosphatase, which enables the export of sucrose.

In the reaction scheme above, G1P is converted to UDP-Glc through the following reaction:

G1P + H⁺ + UTP ↔ UDP-Glc + P_i

This reaction is just mildly favored thermodynamically.

Before studying the enzyme's structure, we will first discuss the regulation of the sucrose synthesis pathway and the general mechanism of glycosyltransferases.

Regulation

Carbon capture in the light reaction of photosynthesis results in the production of sucrose (for transport) and starch synthesis. The resulting product(s) depend on key regulatory steps. Remember the six trioses formed in the Calvin cycle; five are returned into the cycle for the synthesis of ribulose 1,5-bisphosphate, and only one is used to synthesize sucrose and starch. If too much is removed, the cycle slows. If not enough is used for starch and sucrose synthesis, P_i, which is transported into the stroma from the cytoplasm by an important translocator (see Section 20.3), would become depleted.

The key cytosolic regulatory step is catalyzed by fructose-1,6-bisphosphatase (FBPase-1) and a unique plant enzyme, PPi-dependent phosphofructokinase (PP-PFK-1), which catalyzes the reverse reaction, F6P → F1,6BP, in regulatory steps similar to those in glycolysis and gluconeogenesis.

• fructose 1,6-bisphosphatase (FBPase-1) is inhibited by the allosteric modulator fructose 2,6-bisphosphate (F2,6BP)
• PPi-dependent phosphofructokinase (PP-PFK-1) is activated by fructose 2,6-bisphosphate

The coordinate regulation of these two enzymes is shown in Figure \(\PageIndex{4}\).

 

Figure \(\PageIndex{4}\): Regulation of sucrose synthesis at the formation of fructose-6-phosphate

The substrate for the PPi-dependent phosphofructokinase (PP-PFK-1) is PPi, which serves as a phospho-donor. In the plant cytosol, no pyrophosphatase exists to catalyze the cleavage of PP_i into P_i. Also note that Fru-2,6-P₂ itself is synthesized and degraded by the bifunctional enzyme phosphofructokinase 2/fructose-2,6-bisphosphatase, which we studied before in the regulation of the same step in glycolysis/gluconeogenesis.

The levels of F2,6-BP depend on the rate of photosynthesis:

• When photosynthesis is high (in light conditions), [DHAP] and [3PG] increase, which inhibits PFK2, which decreases F26BP, which causes a differential increase in F1,6BPase activity over PP-PFK-1, which increases F6P for sucrose synthesis as well as Pi for the continuation of the light reactions. This allows sucrose synthesis when excess DHAP and 3PG are produced in the light reactions, which makes biological sense.
• When photosynthesis is low (in dark conditions), the same regulations lead to an increase in F2,6BP, which leads to the preferential activation of the glycolytic enzyme PPi-dependent phosphofructokinase-1 (PP-PFK1) and inhibition of the gluconeogenic enzyme fructose 1,6-bisphosphatase (FBPase-1)

We will see later that the main regulatory step in starch synthesis is ADP-glucose pyrophosphorylase. In contrast to the inhibition by 3PG of PFK2, 3PG (which increases in active photosynthesis) activates ADP-glucose pyrophosphorylase while Pi inhibits it. Pi increases when the synthesis of ATP from ADP and Pi (by ATP synthase in the light reaction) slows (such as in darker conditions). If sucrose synthesis slows and sufficient 3PG persists, the activation of ADP-glucose pyrophosphorylase stimulates starch synthesis.

Glycosyltransferases (GTs)

Glycosyltransferases are crucial enzymes, as they play a vital role in synthesizing most of the planet's biomass. They catalyze the transfer of a monosaccharide from a donor that has been activated by the attachment of a nucleotide in the form of a nucleotide sugar (NDP-sugar) or dolichol-(pyro)phosphate sugar to acceptors. These include other sugars, lipids, and proteins, which are glycosylated on alcoholic side chains (Ser, Thr) or amides (Asn). Over 500,000 different GTs are known and deposited in the Carbohydrate-Active enZYmes Database (CAZy database2). Based on sequence and structure, there are over 114 different families. Although they depart significantly in primary sequence, only three major folds are predominant (GT-A, -B, and -C)

As shown in the reaction below, a glycosyl transferase reaction is required to transfer glucose from a donor, such as UDP-glucose, to an acceptor, like fructose, to form sucrose.

NDP-Glc (donor) + F6P (acceptor) → Sucrose-6-P + NDP

Perhaps now is a good time to study their generic reaction mechanisms before progressing to starch synthesis.

About 65% of glycosyltransferase reactions use nucleotide sugars as donors. These enzymes are called Leloir transferases. They are nucleotide-dependent. The activated NDP-sugar donor binds first to the enzyme, followed by the acceptor, to form a ternary complex. A conformational change enables catalysis, facilitating the sequential release of the products. Hence, the enzyme follows a sequential ordered bi-bi mechanism.

The reaction could proceed with either retention or inversion of the anomeric carbon of the NDP-sugar donor. This is illustrated for the reaction of a C1 α-NDP donor monosaccharide with a monosaccharide acceptor to produce the α(1,4) link with retention of configuration or the β(1,4) link with inversion as shown in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Reaction of a donor NDP-monosaccharide and an acceptor monosaccharide with retention or inversion of configuration at the anomeric carbon of the donor

The same stereochemical outcomes can occur in the hydrolysis of acetal bonds by glycosyl hydrolases.

Reactions that proceed with inversion react in an S_N2 reaction, similar to the nucleophilic attack on alkyl halides. The glycosyl transferase that proceeds with inversion makes the attacking nucleophile on the acceptor more nucleophilic by general base catalysis by a deprotonated glutamic or aspartic acid.

The glycosyl transferase that proceeds with the retention of configuration is less understood. Several alternative mechanisms have been proposed for inverting and retaining glycosyltransferase in general, as illustrated in Figure \(\PageIndex{6}\).

Figure \(\PageIndex{6}\): .Proposed glycosyltransferase mechanisms. Schuman et al. PLOS One (2013). doi:10.1371/journal.pone.0071077.g001. Creative Commons Attribution License

They include the following possible mechanisms:

Panel (A): A double displacement mechanism utilizing two inversions with net retention of stereochemistry involving a covalent glycosyl-enzyme intermediate. The individual steps involve inverting via an SN2 process (B).

Panel (B): Inverting Leloir glycosyltransferases promotes a backside nucleophilic attack on C1 by the acceptor from an inline (usually equatorial) position, resulting in inversion of the anomeric bond stereochemistry.

Panel (C): An orthogonal mechanism consisting of nucleophilic attack on C1 by the acceptor concurrent with leaving group loss from a position approximately at right angles to the C1-leaving group axis.

Panel (D): An S_N1 mechanism involving an intermediate with oxocarbenium character, followed by rapid internal nucleophilic attack by the acceptor nucleophile; or

Panel (E): An S_N1 mechanism involving a discrete oxocarbenium intermediate. All mechanisms require proton transfers of the hydroxyl hydrogen of the acceptor to an enzymatic base or the departing leaving group.

From your chemistry studies, you will remember that S_N1 reactions are dissociative and form a positively charged carbocation intermediate. In an S_N-1 reaction, the intermediate exhibits cationic character but is not fully charged. In the case of glycosyltransferases, the intermediates would be oxycarbenium ions. (Carbenium ion can be considered carbocations with three bonds to the carbon). The S_N1 reaction will occur only if the formation of the ion is activated and the ion is stabilized. A protic solvent is typically required for stabilization if the reaction occurs in solution. An appropriate arrangement of negatively or partially negatively charged atoms in the backbone and side chains is required to stabilize S_N1 mechanisms in the enzyme's anhydrous active site.

The double replacement reactions (Panel A) require a side-chain nucleophile, and likely candidates for retaining glycosyltransferases are not positioned for this task. The evidence suggests an orthogonal mechanism. It appears that the binding of the donor is similar in retaining transferases, such that it is in a 90⁰ position of nucleophilic attack by the acceptor, which leads to a trigonal bipyramidal transition state with the nucleophile axial and the leaving group equatorial (orthogonal).

The donor NDP-monosaccharides typically are Mn²-containing proteins with the inverting and retaining transferase having different coordination geometries for Mn²⁺ binding, as illustrated in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Coordination geometries for Mn²⁺ binding in glycosyltransferase. Shuman, ibid.

Inverting enzymes such as GalT1 (top) achieve nearly perfect octahedral geometry about the coordinated metal ion (displayed angles of 81° and 91°, compared to the ideal octahedral 90° bond angles), with the acceptor nucleophile subsequently placed “inline” (approaching 180°) for a classic inverting S_N2 backside attack. Retaining enzymes, such as GTA (bottom), however, utilize an arrangement of acidic residues, often with acute bidentate Asp coordination, which severely skews the metal geometry (displayed angles of 54° and 115°) and allows sufficient room between phosphate oxygens for orthogonal attack from the acceptor. U is uridine, C1 is the donor galactose C1.

Figure \(\PageIndex{8}\) shows a possible mechanism for transferring a monosaccharide from the donor ADP-sugar through an oxycarbenium intermediate to an acceptor.

Figure \(\PageIndex{8}\): Possible mechanism for the transfer of a monosaccharide from the donor ADP-sugar through an oxycarbenium intermediate to an acceptor (example - a growing starch chain). Schuman B, Evans SV, Fyles TM (2013) Geometric Attributes of Retaining Glycosyltransferase Enzymes Favor an Orthogonal Mechanism. PLoS ONE 8(8): e71077. doi:10.1371/journal.pone.0071077. Creative Commons Attribution License

As mentioned above, glycosyltransferases are classified into three major folds: GT-A, GT-B, and GT-C. Different representations of the GT-A fold core structure predicted through analysis by neural networks and deep learning are shown in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\): Fold core of GT-As. Taujale, R., Zhou, Z., Yeung, W. et al. Mapping the glycosyltransferase fold landscape using interpretable deep learning. Nat Commun 12, 5656 (2021). https://doi.org/10.1038/s41467-021-25975-9. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

This computational modeling of structure utilizes simple secondary structure representations derived from primary sequences to predict folds independently of the sequence. GT folds are accurately predicted by learning secondary-structure features without primary-sequence alignment.

Panel (a) shows a linear map of the conserved secondary core structures (below) and graphs (top). The blue line represents a conservation score, and the red is a CAM score. CAM values correspond to residue positions that distinguish them the most from other class labels (folds and families).

Panel (b) shows the core's structural alignment with the CAM values mapped onto it. The conserved regions have a high CAM value, indicated by a high green intensity and a low purple intensity.

Panel (c) shows the consensus secondary structure on the left for the aligned positions in the two GT-A fold clusters (blue: beta-sheets; red: helices; green: loops). Average CAM values from different "layers" of analysis are shown for each aligned position (the higher the green intensity, the higher the CAM value). Cyan and magenta boxes highlight the secondary structure differences between the two clusters near the hypervariable HV2 and HV3 regions, respectively. The conserved DXD motif, G-loop, and C-His are indicated for reference. Donor and acceptor substrates for GT-A0 are shown as sticks.

This figure provides readers with a sense of the complexity of the analysis required to understand and differentiate the structure-function features of these structurally similar but complex enzymes.

Structure and Enzymatic Activity of Sucrose Synthase (SuSy)

These enzymes are usually homotetramers with a monomeric molecular weight of around 90,000. The monomers typically have an N-terminal domain that directs the enzyme's targeting to a specific location and a C-terminal GT-B domain. It can be regulated by phosphorylation at a serine near position 12 in the N-terminal domain, which presumably affects its cellular localization, and another near position 170, which impacts its degradation. Two glutamates in the C-terminal GT-B domain (E678 and E686) and phenylalanine (680) are essential for the catalytic activity. Sucrose synthase is reversible, as is the synthesis of sucrose-6-P from F6P and UDP-glucose, which can be reversed in the presence of UDP. The enzyme can also use ADP-glucose as a donor.

The structure of the Thermosynechococcus elongatus sucrose phosphate synthase with bound UDP and sucrose-6-Phosphate has been solved and along with other studies a reaction mechanism proposed as shown in Figure \(\PageIndex{10}\).

Figure \(\PageIndex{10}\): Catalytic model of TeSPS. Yuying et al. Co-crystal Structure of Thermosynechococcus elongatus Sucrose Phosphate Synthase With UDP and Sucrose-6-Phosphate Provides Insight Into Its Mechanism of Action Involving an Oxocarbenium Ion and the Glycosidic Bond. Frontiers in Microbiology, 11, 2020. https://www.frontiersin.org/article/...icb.2020.01050. Creative Commons Attribution License (CC BY).

(A)The state before the reaction is shown. (B)The glucose residue of UDPG forms hydrogen bonds between/among the phosphate groups, His158, Glu331, and F6P. Due to the formation of these hydrogen bonds, the pyranose ring of the glucose becomes negatively charged to promote C1 to form an oxocarbenium ion. (C)The relatively weak hydrogen bond formed by His158 and O6 is broken, which causes the pyranose ring to lose some negative charge character and forces the C1 oxocarbenium ion to form a covalent bond with the F6P oxygen atom. (D)UDP and S6P are released from the catalytic center.

A 2D view of the active site residues is shown in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\). The catalytic center of TeSPS. Yuying et al. ibid.

The catalytic center of TeSPS. Loops 1, 3, 4, and 5 form a cave that binds to the uracil moiety of UDP. Glu339 stabilizes the ribose ring through two hydrogen bonds. Leu335 forces two phosphate groups in UDP to reorient. Several basic amino acids, including Arg105, Arg178, Arg249, and Arg253, interact with the phosphate groups of UDP and S6P via ionic bonds. Pro332 at the turn of loop 6 interacts with the pyranose ring via CH/π bonds. All hydroxyl groups (O2, O3, O4, and O6) of the glucose moiety of S6P form hydrogen bonds with phosphate groups or the side chains of various amino acids. “O2” forms a hydrogen bond with P1O1 of the P1 phosphate group of UDP. “O3” forms a hydrogen bond with the carboxyl group of Glu331. “O4” forms a hydrogen bond with P2O1 of the P2 phosphate group of UDP. “O6” forms a hydrogen bond with the imidazole side chain of His158. The distances between groups are indicated in the figure.

Figure \(\PageIndex{12}\) shows an interactive iCn3D model of the Thermosynechococcus elongatus Sucrose Phosphate Synthase With UDP and Sucrose-6-Phosphate (6KIH)

Figure \(\PageIndex{12}\): Thermosynechococcus elongatus Sucrose Phosphate Synthase With UDP and Sucrose-6-Phosphate (6KIH) (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...1kCnQ9pxcJjdy7

Metabolism of Sucrose

Sucrose is a primary synthesis product of photosynthesis and is transported to other plant "sink" tissues where it is used for both energy and biosynthetic precursors. Suc transporters can move it from the phloem to the apoplast. It can enter sink cells via sucrose transporters or be hydrolyzed by cell wall invertase (cwINV) to yield glucose (Glc) and fructose (Fru), which enter the cells via hexose transporters. Suc can also pass directly from the phloem to sink cells through plasmodesmata (physical connections between cells). Inside sink cells, sucrose (Suc) can be metabolized or transported to the vacuole, where it can be stored as sucrose, transformed into fructans by fructosyltransferases (FTs), or hydrolyzed by vacuolar invertase (vINV) and stored as hexoses. To be metabolized, Suc must be hydrolyzed by either cytosolic invertase (INV) into glucose and fructose or by the reversible reaction of sucrose synthase (SuSy) using UDP instead of water to yield fructose and UDP-G. These processes are illustrated in Figure \(\PageIndex{13}\).

 

Figure \(\PageIndex{13}\): Simplified schematic presentation of sugar metabolism in sink tissue cells toward cellulose, callose, and starch synthesis. Stein and Granot. Frontiers in Plant Science (10). 2019. https://www.frontiersin.org/article/...pls.2019.00095. DOI=10.3389/fpls.2019.00095 https://www.frontiersin.org/articles...019.00095/full

The hexoses (glucose and fructose) can be phosphorylated to hexose phosphates (hex-P), directed to starch synthesis in the plastid or glycolysis, and then respiration in the mitochondria or directed to other metabolic pathways. Plasma membrane-associated SuSy (pmSuSy) and cwSUS can generate UDP-G, which is used to synthesize cellulose for cell walls and callose for plugging plasmodesmata. Callose is a polysaccharide in the form of β-1,3-glucan with some β-1,6-branches in the cell walls of a wide variety of higher plants.

Biosynthesis of Starch

During active photosynthesis in bright light, a plant leaf produces more carbohydrates (as triose phosphates) than it needs to generate energy or synthesize precursors. The excess is converted into sucrose and transported to other plant parts for storage or use as fuel. In most plants, starch is the primary storage form, but in a few plants, such as sugar beet and sugarcane, sucrose is the primary form. The synthesis of sucrose and starch occurs in different cellular compartments (the cytosol and plastids, respectively), and these processes are coordinated by various regulatory mechanisms that respond to changes in light levels and photosynthetic rates.

Starch Synthesis

Starch, like glycogen, is a homopolymer of D-glucose in (α1,4) linkage with (α1,6) branches. Glycogen is found in all three domains of life: Archaea, Bacteria, and Eukarya. In contrast, starch is found in photosynthetic algae, land plants, and some cyanobacterial species.

Starch is synthesized by starch synthase in chloroplasts for temporary storage and in amyloplasts in seeds, roots, and underground stems (such as tubers) for long-term storage. As with the synthesis of glycogen, the glycosyltransferase catalyzes the addition of an activated ADP-glucose to the acceptor, the elongating starch polymer.

ADP-α-D-glucose-1-phosphate (donor) + [(1→4)-α-D-glucosyl]_(n) (acceptor) ↔ [(1→4)-α-D-glucosyl]_(n+1) + ADP + H⁺

The ADP-glucose donor is formed in the following reaction:

Glucose 1-phosphate + ATP ↔ ADP-glucose + PPi

There is a pyrophosphatase in plastids (but not in the cytosol), making the reaction irreversible. Hence, the next overall reaction is

Starch_n + glucose 1-phosphate + ATP → starch_{n + 1} + ADP + 2Pi

Taking into account the hydrolysis by inorganic pyrophosphatase of the PPi produced during ADP-glucose synthesis, the overall reaction for starch formation from glucose 1-phosphate is:

Starch_n + glucose 1-phosphate + ATP → starch_{n + 1} + ADP + 2Pi; ΔG'º = -50 kJ/mol = -12 kcal/mole (-50 kJ/mol)

ΔG'º = -50 kJ/mol = -12 kcal/mol

In glycogen synthesis, the donor is UDP-glucose, which is added to the reducing end (C1) of the growing glycogen polymer (with the C1 OH acting as a nucleophile), so the polymer extends from that end. Kinetic models suggest that starch synthases, which use ADP-glucose, may use two different active sites that appear to alternately add glucose to the nonreducing C4 end (with the C4 OH acting as a nucleophile), with the reducing end of the linear α(1,4) polymer being alternately covalently attached to one site, then the other, with the attachment activating that end for reaction with C4-OH of the polymer at the other site.

Figure \(\PageIndex{14}\) shows a very simplified structure of the starch synthase using two different active sites as the reaction proceeds through the first steps.

Figure \(\PageIndex{14}\) shows a simplified structure of the starch synthase using two active sites

Note that additional glucose units are added to the nonreducing C4 end, denoted by a star. α(1,6) branches are added by branching enzymes, as in the case of glycogen. Bacterial starch is made like glycogen but uses ADP-glucose, as does plant starch synthase. Structural models show that the protein has one active site, so the kinetic models suggesting the use of two active sites may refer to a movement of chains between different monomers in oligomeric forms of the protein.

Both the donor (ADP-Glc) and product (starch_n+1) have their participating electrophile (ADP) and now-substituted nucleophile (C4-O-R) in the α-anomeric form, so they are retaining glycosyltransferases. The enzymes could act in two different ways:

• a processive mechanism: the acceptor (starch_n) stays bound to the enzyme after each addition of the next glucose from the donor ADP-glc, and one chain extends quickly
• a distributive mechanism: the enzyme dissociates from the product (starch_n+1) after the addition of the glucose from ADP-glc and must rebind to catalyze the next addition, so many new chains start, and the growth of each chain is slow

Kinetic evidence suggests that some starch synthases are processive and others are distributive. Along with starch synthase, three other enzymes are involved: ADP-glucose pyrophosphorylase (AGPase), starch branching enzyme (SBE), and starch debranching enzyme (DBE)

Starch synthesis is regulated by gene transcription, phosphorylation, and redox conditions. A key regulatory enzyme is an ADP-glucose pyrophosphorylase (AGPase). This enzyme catalyzes the formation of ADP-glucose (the donor) and PPi from glucose 1-phosphate and ATP. Dithiothreitol, a reducing agent, increases starch synthesis by inactivating AGPase. We will see below that a key disulfide bond is present in some starch synthases that must be reduced (cleaved) to open an active site cleft between the N-terminal and C-terminal lobes of the catalytic domain. In vivo, thioredoxins are probably involved in redox regulation.

Structures of starch synthetases (SS)

There are four classes of soluble starch synthetases (SSI-SSIV) and one starch granule-bound one (GBSS). As noted in the mechanism above, all have 2 catalytic domains, except SSII, which has 3 CHO-binding domains. GBSS appears to form amylose and long chains of amylopectins (amylose with around 5% α(1,6) branching. Loss-of-function mutants of GBSS have much-reduced amylose concentrations. SSI-SSIII produces most of the amylopectin. SSI is most active with chains (the outer ones in branched structures) with around 7-9 glucose units. In leaves, it generates chains of up to 10 residues in amylopectin. The SSs can be chloroplastic or amyloplastic.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of barley starch synthase I in complex with maltooligosaccharide (4HLN).

Figure \(\PageIndex{15}\): Barley starch synthase I in complex with maltooligosaccharide (4HLN). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...LGwFUeJAMskBcA

The protein is shown in gray, in an open conformation, with a 5-mer of glucose bound on the outside. It has a GT-B fold with distinct N- and C-terminal Rossmann-like domains and a central linker. Side chains within 5 Å of the oligosaccharide are shown in sticks and labeled. Note especially the disulfide bridge between cysteines 126 and 506 in the central region of the protein, which prevents formation of the active site. This clearly shows the importance of redox signaling in activating the enzyme.

The maltose is not bound in the active site but at a surface secondary binding site (SBS). The role of this site is somewhat unclear, but it may be involved in carbohydrate-carbohydrate interactions. Specifically, they may assist in recruiting starch chains for further elongation. SBSs are found in many but not all starch synthases. Note that in this structure, there is only one occluded active site and not two, as suggested in Figure 14 above.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of the catalytic domain of starch synthase IV from Arabidopsis thaliana bound to ADP and acarbose (6GNE).

Figure \(\PageIndex{16}\): Catalytic domain of starch synthase IV from Arabidopsis thaliana bound to ADP and acarbose (6GNE) https://structure.ncbi.nlm.nih.gov/i...MioUaKM73M3Wd9

This structure is just the catalytic domain (representing about half of the total protein sequence). The N-terminal domain is colored by secondary structure. Acarbose (spacefill, CPK colors) again occupies both the donor and acceptor sites in the active site (central regions). The structure has a secondary binding site (SBS) occupied by the disaccharide maltose. Again, there are not two active sites, but the migration of starch chains between monomers in oligomeric forms could support the model shown in Figure 14.

The structure of beta-acarbose, an inhibitor, is shown in Figure \(\PageIndex{17}\).

Figure \(\PageIndex{17}\): structure of beta-acarbose

Figure \(\PageIndex{x18}\) below shows an interactive iCn3D model of the Granule Bound Starch Synthase from Cyanobacterium sp. CLg1 bound to acarbose and ADP (6GNF)

Figure \(\PageIndex{18}\): Granule Bound Starch Synthase from Cyanobacterium sp. CLg1 bound to beta-acarbose and ADP (6GNF). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?F1tJBDAh6QRhxpH88

ADP, which occupies part of the donor site that would usually bind NTP-Glc, is shown in CPK-colored sticks. Beta-acarbose occupies the rest of the donor site (where Glc of NTP-Glc would bind) and the acceptor site (growing starch chain). It is shown in the CPK-colored wire-frame surface. Histidine 181, which probably stabilizes an " oxocarbenium-like anomeric carbon in the transition state," is shown in balls and sticks with CPK colors.

Cellulose Synthesis

We can't leave plant carbohydrate metabolism without considering cellulose synthesis, which is catalyzed in plants by members of the superfamily cellulose synthase (CesA) and cellulose synthase-like (CsI) enzymes, both of which are part of the glycosyltransferase GT2 family and share similar structures. Cellulose and hemicellulose are the chief components of the 1⁰ and 2⁰ cell walls. Members of the CesA family have a conserved motif (DDDQxxRW) and a zinc-finger domain. Different members catalyze the synthesis of the 1⁰ and 2⁰ cell walls. Members of the Csl family are involved in additional cell wall glycans, including (1,4)-β-D-mannan (CsIA) and xyloglucan cytoskeleton (CslC). UDP-glucose is the donor in creating the β(1,4) acetal linkages between glucose monomers.

Plant growth must respond to environmental triggers by balancing cell expansion and cell division. A key regulator of these processes is the cell wall's flexibility, which can maintain turgor pressure through expansion. Nonexpanding cells (for example, those that line the xylem vessels and are in woody tissue) have secondary cell walls beneath their primary walls.

We have previously discussed the primary and secondary cell wall structure in Chapter 7.3. In brief, the primary cell walls contain cellulose, hemicellulose, and pectins. Cellulose, the main component that provides strength, is synthesized by CesA, which forms a very large rosette-shaped complex (CSC). These complexes appear to regulate the movement of intracellular microtubules, which guide the synthase complex through interactions with microtubule-associated cellulose synthase compartments (MASCs), whose numbers increase during stress. Likewise, there are uncoupling proteins that inhibit microtubule movement from the CSC. These protein complexes stay aligned during cell growth. Hence, the cell wall plays a key role in signal transduction, facilitating growth and cell division.

The structure of the rosette-shaped complex (CSC) has been determined by cryoEM and is shown in Figure \(\PageIndex{19}\).

Figure \(\PageIndex{19}\): Structural cartoons of the CESA CSC complex. Nixon, B., Mansouri, K., Singh, A., et al. Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex. Sci Rep 6, 28696 (2016). https://doi.org/10.1038/srep28696. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/

Panel (A) shows the complex as a series of trimers of CESAs, with each monomer spanning the membrane with 7 alpha helices. The catalytic domain is in the cytoplasm.

Panel (B) shows the CSC complex with the top leaflet removed

Panel (C) shows a top-down view of 6 sets of trimers of CESA.

Panel (D) shows a metal replica viewed in the TEM after the biological material has been removed.

Each trimer synthesizes a cellulose strand. There are 18 trimers in the complex, allowing the concomitant synthesis of cellulose strands that can easily self-associate through hydrogen bonding to form near the extracellular surface cellulose protofibrils.Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the catalytically active homotrimeric poplar cellulose synthase (6WLB)

Figure \(\PageIndex{20}\): Catalytically active homotrimeric poplar cellulose synthase (6WLB). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...zCm6fYSwAFA897

Each monomer in the trimers is given a different color. A 5-residue β(1,4) glycan is shown in cartoon form emerging into the middle of the membrane complex.

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the homotrimeric poplar cellulose synthase isoform glycan binding site (6WLB).

Figure \(\PageIndex{21}\): Homotrimeric poplar cellulose synthase isoform glycan binding site (6WLB. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...HqYhTQfuw857PA

The actual transmembrane channels start just above the active site. Key amino acid side chains (Trp 718, Phe 513, Val 529, and Gln 494) help form the portal opening. The actual channel is lined with both aromatic and hydrophilic residues, which provide sufficient but not overly strong noncovalent interactions that allow the continually synthesized cellulose to move in a sequential manner as it ratchets forward toward the extracellular side of the membrane. The aromatic residues interact with the glucose residues via π-stacking and with the equatorial OH groups on the β-glucose polymer. Remember that cellulose is especially stable from a steric perspective since all its OH groups and the acetal linkage are equatorial.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter describes the biosynthesis of the major plant carbohydrates — sucrose, starch, and cellulose — beginning with the triose phosphates produced by the Calvin cycle, and integrating mechanistic enzymology, structural biology, and regulatory biochemistry throughout.

Sucrose is the primary transport form of photosynthetically fixed carbon in plants, fulfilling a role analogous to ketone bodies in animal lipid transport: it is a stable, water-soluble form of chemical energy that can move from source tissues (leaves) to sink tissues (roots, fruits, seeds) via the phloem. Its chemical stability arises from its unique disaccharide linkage: the acetal bond connects the anomeric C1 of glucose to the anomeric C2 of fructose, creating a fully protected (non-reducing) sugar that cannot open to form a reactive aldehyde. This prevents sucrose from glycating proteins (unlike glucose, which can react with hemoglobin's amino groups via a Schiff base mechanism to form HbA1c, a diabetes marker), resists amylase cleavage, and makes it an ideal transport molecule. Sucrose is synthesized in the cytosol: glucose-1-phosphate is converted to UDP-glucose by UTP-glucose-1-phosphate uridylyltransferase; sucrose-6-phosphate synthase then transfers the glucosyl group from UDP-glucose to fructose-6-phosphate to form sucrose-6-phosphate; and sucrose-6-phosphatase hydrolyzes the product to free sucrose for export.

Sucrose synthesis is regulated at the fructose-1,6-bisphosphate/fructose-6-phosphate interconversion step through the reciprocal regulation of cytosolic fructose-1,6-bisphosphatase (FBPase-1, inhibited by fructose-2,6-bisphosphate, F2,6BP) and PPi-dependent phosphofructokinase (PP-PFK-1, activated by F2,6BP). F2,6BP levels are controlled by the bifunctional PFK2/FBPase-2 enzyme, whose kinase activity is inhibited by elevated DHAP and 3PG (which accumulate during active light reactions). In bright light, high DHAP and 3PG suppress PFK2, lower F2,6BP, and favor FBPase-1, increasing fructose-6-phosphate for sucrose synthesis and freeing Pi for return to the chloroplast. In the dark, the opposite regulation activates glycolysis to provide energy. Starch synthesis is coordinately regulated by 3-phosphoglycerate activating and Pi inhibiting ADP-glucose pyrophosphorylase — so excess carbon during active photosynthesis promotes both sucrose and starch synthesis, while declining light conditions shut down synthesis of both.

The enzymes that build the glycosidic bonds of sucrose, starch, and cellulose are all members of the glycosyltransferase (GT) superfamily, the most diverse enzyme family known (~500,000 members in three major fold families GT-A, GT-B, and GT-C). Glycosyltransferases catalyze the transfer of activated sugar donors (NDP-sugars, most commonly UDP-glucose or ADP-glucose) to acceptor molecules via either an inverting or a retaining mechanism. Inverting transferases use Mn²⁺ in a near-perfect octahedral geometry to facilitate an inline S_N2 backside nucleophilic attack, with a deprotonated Glu or Asp acting as a general base. Retaining transferases use Mn²⁺ in a distorted geometry, with an acute bidentate aspartate ligand that opens space for orthogonal nucleophilic attack by the acceptor, proceeding through an oxocarbenium-like transition state rather than a classical S_N2 or double-displacement mechanism. All three reactions studied in this chapter — sucrose phosphate synthase, starch synthase, and cellulose synthase — employ retaining mechanisms producing α or β products via oxocarbenium-like transition states, with conserved His, Glu, Asp, and Arg residues stabilizing the charged intermediate.

Starch, like glycogen, is a polymer of glucose in α(1,4) linkage with α(1,6) branches, but differs in using ADP-glucose as the activated donor rather than UDP-glucose, and in extending the chain at the nonreducing end through a proposed two-active-site mechanism in which successive glucose additions involve migration of the growing chain between alternative sites on the enzyme (or between monomers in oligomeric enzyme forms). The key regulated enzyme is ADP-glucose pyrophosphorylase (AGPase), which synthesizes the ADP-glucose donor; the reaction is driven forward by the hydrolysis of PPi by plastidic pyrophosphatase. Starch synthases are additionally regulated by redox: a disulfide bond between conserved cysteines in SSI closes the active site cleft between the N- and C-terminal Rossmann domains, and thioredoxin-mediated reduction of this disulfide (powered by the ferredoxin-thioredoxin reductase system activated by light reactions) opens the active site and activates starch synthesis. Multiple starch synthase isoforms (SSI–SSIV and GBSS) contribute different chain lengths and architecture to amylopectin and amylose synthesis, with branching enzymes adding α(1,6) linkages and debranching enzymes trimming inappropriate branches.

Cellulose synthesis represents a fundamentally different structural context: cellulose is a linear β(1,4)-linked glucan polymer in which all hydroxyl groups and the acetal oxygen are equatorial, permitting extensive intermolecular hydrogen bonding and producing an extremely rigid, water-insoluble fiber that constitutes the primary structural component of plant cell walls. Cellulose is synthesized by cellulose synthase (CesA), a GT-B family enzyme with a conserved DDDQxxRW motif and zinc-finger domain, that assembles into a large rosette-shaped cellulose synthase complex (CSC) of 18 CesA trimers arranged symmetrically in the plasma membrane. Each trimer synthesizes one glucan chain, threading it through a transmembrane channel lined with aromatic residues (which provide π-stacking interactions with the glucose rings) and hydrophilic residues (which interact with equatorial OH groups), allowing the growing cellulose chain to ratchet forward toward the extracellular surface. Concomitant synthesis of 18 glucan chains by the CSC enables their immediate lateral self-association into cellulose protofibrils through hydrogen bonding. The CSC's movement through the plasma membrane is guided by cortical microtubules through microtubule-associated cellulose synthase compartments (MASCs), linking cellulose deposition to the cell's cytoskeletal architecture and thereby coupling cell wall synthesis to growth direction and cell division.