Synthesis of Cell Wall Polysaccharides: Plant Cellulose and Bacterial Peptidoglycan:- Cellulose Is Synthesized by Supramolecular Structures in the Plasma Membrane
The complex enzymatic machinery that assembles cellulose chains spans the plasma membrane, with one part positioned to bind the substrate, UDP-glucose, in the cytosol and another part extending to the outside, re sponsible for elongating and crystallizing cellulose molecules in the extracellular space. Freeze-fracture electron microscopy shows these terminal complexes, also called rosettes, to be composed of six large particles arranged in a regular hexagon (Fig. 20–30). Several proteins, including the catalytic subunit of cellulose synthase, make up the terminal complex. Cellulose synthase has not been isolated in its active form, but its amino acid sequence has been determined from the nucleotide sequence of the gene that encodes it. From the primary structure we can use hydropathy plots (see Fig. 11–11) to deduce that the enzyme has eight trans membrane segments, connected by short loops on the outside, and several longer loops exposed to the cytosol. Much of the recent progress in understanding cellulose synthesis stems from genetic and molecular genetic studies of the plant Arabidopsis thaliana, which is especially amenable to genetic dissection and whose genome has been sequenced.
FIGURE 20–30 Rosettes. The outside surface of the plant plasma mem brane in a freeze-fractured sample, viewed here with electron microscopy, contains many hexagonal arrays of particles about 10 nm in diameter, believed to be composed of cellulose synthase molecules and associated enzymes.
New cellulose chains appear to be initiated by the formation of a lipid-linked intermediate unlike anything involved in starch or glycogen synthesis. Glucose is trans ferred from UDP-glucose to a membrane lipid, probably the plant sterol sitosterol (Fig. 20–31), on the inner face of the plasma membrane. Here, intracellular cellulose synthase adds several more glucose residues to the first one, in (β 1→4) linkage, forming a short oligosaccharide chain attached to the sitosterol (sitosterol dextrin). Next, the whole sitosterol dextrin flips across to the outer face of the plasma membrane, where most of the polysac charide chain is removed by endo-1,4-β-glucanase. The shortened sitosterol dextrin primer now associates, perhaps covalently, with another form of cellulose synthase. Presumably this entire process occurs in the rosettes. Whether each of the 36 cellulose chains is initiated on its own lipid primer, or the primer recycles to start a number of chains, is not yet clear. In either case, the second form of cellulose synthase extends the polymer to 500 to 15,000 glucose units, extruding it onto the outer surface of the cell. The action of the enzyme is processive: one enzyme molecule adds many glucose units before re leasing the growing cellulose chain. The direction of chain growth (whether addition occurs at the reducing end or at the nonreducing end) has not been established. The finished cellulose is in the form of crystalline microfibrils (Fig. 20–29), each consisting of 36 separate cellulose chains lying side by side, all with the same (parallel) orientation of nonreducing and reducing ends. It seems likely that each particle in the rosette synthesizes six separate cellulose chains simultaneously and in parallel with the chains made by the other five particles, so that 36 polymers arrive together on the outer surface of the cell, already aligned and ready to crystallize as a microfibril of the cell wall. When the 36 polymers reach some critical length, their synthesis is terminated by an unknown mechanism; crystallization into a microfibril follows. In addition to its catalytic subunit, cellulose synthase may have subunits that mediate extrusion of the polysaccharide chain (the pore subunit) and crystallization of the polysaccharide chains outside the cell (the crystallization subunit). The potent herbicide CGA 325615, which specifically inhibits cellulose synthesis, causes rosettes to fall apart; the small amount of cellulose still synthesized remains tightly, perhaps covalently, bound to the catalytic subunit of cellulose synthase. The inhibitor may act by dissociating the catalytic subunit from the pore and crystallization subunits, preventing the later stages of cellulose synthesis. The UDP-glucose used for cellulose synthesis is generated from sucrose produced during photosynthesis, by the reaction catalyzed by sucrose synthase (named for the reverse reaction):
Sucrose+ UDP→ UDP- glucose+ fructose
In one proposed model, cellulose synthase spans the plasma membrane and uses cytosolic UDP-glucose as the precursor for extracellular cellulose synthesis. In another, a membrane-bound form of sucrose synthase forms a complex with cellulose synthase, feeding UDP-glucose from sucrose directly into cell wall synthesis (Fig. 20–32). In the activated precursor of cellulose (UDP glucose), the glucose is -linked to the nucleotide, but in the product (cellulose), glucose residues are β (1→4) linked, so there is an inversion of configuration at the anomeric carbon (C-1) as the glycosidic bond forms. Glycosyltransferases that invert configuration are generally assumed to use a single-displacement mechanism, with nucleophilic attack by the acceptor species at the anomeric carbon of the donor sugar (UDP-glucose). Certain bacteria (Acetobacter, Agrobacteria, Rhizobia, and Sarcina) and many simple eukaryotes also carry out cellulose synthesis, apparently by a mechanism similar to that in plants. If the bacteria use a mem brane lipid to initiate new chains, it cannot be a sterol— bacteria do not contain sterols.
FIGURE 20–31 Lipid primer for cellulose synthesis. This proposed pathway begins with 1 the transfer of a glucosyl residue from UDP glucose to a lipid “primer” (probably sitosterol) in the inner leaflet of the plasma membrane. After this initiation, 2 the chain of carbohydrate is elongated by transfer of glucosyl residues from UDP-glucose, until 3 a critical length of oligosaccharide is reached. 4 The sitosterol with its attached oligosaccharide now flips from the inner leaflet to the outer leaflet. 5 An endo-1,4--glucanase separates the growing chain from a short oligonucleotide still attached to the lipid. As it is pushed out of the cell, 6 the lipid-free polymer of glucosyl residues (the glucan acceptor) is further extended by the addition of glucosyl residues from UDP-glucose, catalyzed by cellulose synthase. 7 The lipid-linked oligosaccharide returns to serve as the primer for another chain of cellulose.
FIGURE 20–32 A plausible model for the structure of cellulose synthase. The enzyme complex includes a catalytic subunit with eight transmembrane segments and several other subunits that are presumed to act in threading cellulose chains through the catalytic site and out of the cell, and in the crystallization of 36 cellulose strands into the paracrystalline microfibrils shown in Figure 20–29.
