In vitro function, assembly, and interaction of primary cell wall cellulose synthase homotrimers.

Plant cell walls contain a meshwork of cellulose fibers embedded into a matrix of other carbohydrate and non-carbohydrate-based biopolymers. This composite material exhibits extraordinary properties, from stretchable and pliable cell boundaries to solid protective shells. Cellulose, a linear glucose polymer, is synthesized and secreted across the plasma membrane by cellulose synthase (CesA). Plants express several CesA isoforms, with different subsets necessary for primary and secondary cell wall biogenesis. The produced cellulose chains can be organized into fibrillar structures and fibrillogenesis likely requires the supramolecular organization of CesAs into pseudo sixfold symmetric complexes (CSCs). Here, we structurally and functionally characterize a set of soybean (Gm) CesA isoforms implicated in primary cell wall biogenesis. Cryogenic electron microscopy analyses of catalytically active GmCesA1, GmCesA3, and GmCesA6 reveal their assembly into homotrimeric complexes, stabilized by a cytosolic plant conserved region. Contrasting secondary cell wall CesAs, a peripheral position of the C-terminal transmembrane helix creates a large, lipid-exposed lateral opening of the enzymes' cellulose-conducting transmembrane channels. Co-purification experiments reveal that homotrimers of different CesA isoforms interact in vitro and that this interaction is independent of the enzymes' N-terminal cytosolic domains. Our data suggest that cross-isoform interactions are mediated by the class-specific region, which forms a hook-shaped protrusion of the catalytic domain at the cytosolic water-lipid interface. Further, inter-isoform interactions lead to synergistic catalytic activity, suggesting increased cellulose biosynthesis upon homotrimer interaction. Combined, our structural and biochemical data favor a model by which homotrimers of different CesA isoforms assemble into a microfibril-producing CSC.

Mechanism of mixed-linkage glucan biosynthesis by barley cellulose synthase-like CslF6 (1,3;1,4)-ß-glucan synthase.

Mixed-linkage (1,3;1,4)-ß-glucans, which are widely distributed in cell walls of the grasses, are linear glucose polymers containing predominantly (1,4)-ß-linked glucosyl units interspersed with single (1,3)-ß-linked glucosyl units. Their distribution in cereal grains and unique structures are important determinants of dietary fibers that are beneficial to human health. We demonstrate that the barley cellulose synthase-like CslF6 enzyme is sufficient to synthesize a high-molecular weight (1,3;1,4)-ß-glucan in vitro. Biochemical and cryo-electron microscopy analyses suggest that CslF6 functions as a monomer. A conserved "switch motif" at the entrance of the enzyme's transmembrane channel is critical to generate (1,3)-linkages. There, a single-point mutation markedly reduces (1,3)-linkage formation, resulting in the synthesis of cellulosic polysaccharides. Our results suggest that CslF6 monitors the orientation of the nascent polysaccharide's second or third glucosyl unit. Register-dependent interactions with these glucosyl residues reposition the polymer's terminal glucosyl unit to form either a (1,3)- or (1,4)-ß-linkage.

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales).

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

Architecture of a catalytically active homotrimeric plant cellulose synthase complex.

Cellulose is an essential plant cell wall component and represents the most abundant biopolymer on Earth. Supramolecular plant cellulose synthase complexes organize multiple linear glucose polymers into microfibrils as load-bearing wall components. We determined the structure of a poplar cellulose synthase CesA homotrimer that suggests a molecular basis for cellulose microfibril formation. This complex, stabilized by cytosolic plant-conserved regions and helical exchange within the transmembrane segments, forms three channels occupied by nascent cellulose polymers. Secretion steers the polymers toward a common exit point, which could facilitate protofibril formation. CesA's N-terminal domains assemble into a cytosolic stalk that interacts with a microtubule-tethering protein and may thus be involved in CesA localization. Our data suggest how cellulose synthase complexes assemble and provide the molecular basis for plant cell wall engineering.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase.

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual ß-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize ß-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

Insights into the structure and function of membrane-integrated processive glycosyltransferases.

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. Here, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin, alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).

Biotechnological approaches for field applications of chitooligosaccharides (COS) to induce innate immunity in plants.

Plants have evolved mechanisms to recognize a wide range of pathogen-derived molecules and to express induced resistance against pathogen attack. Exploitation of induced resistance, by application of novel bioactive elicitors, is an attractive alternative for crop protection. Chitooligosaccharide (COS) elicitors, released during plant fungal interactions, induce plant defenses upon recognition. Detailed analyses of structure/function relationships of bioactive chitosans as well as recent progress towards understanding the mechanism of COS sensing in plants through the identification and characterization of their cognate receptors have generated fresh impetus for approaches that would induce innate immunity in plants. These progresses combined with the application of chitin/chitosan/COS in disease management are reviewed here. In considering the field application of COS, however, efficient and large-scale production of desired COS is a challenging task. The available methods, including chemical or enzymatic hydrolysis and chemical or biotechnological synthesis to produce COS, are also reviewed.

Oligomerization, conformational stability and thermal unfolding of Harpin, HrpZPss and its hypersensitive response-inducing c-terminal fragment, C-214-HrpZPss.

HrpZ-a harpin from Pseudomonas syringae-is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (∼10.0 kcal/mol) of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities.

Bacterial chitin binding proteins show differential substrate binding and synergy with chitinases.

Glycosyl hydrolase (GH) family 18 chitinases (Chi) and family 33 chitin binding proteins (CBPs) from Bacillus thuringiensis serovar kurstaki (BtChi and BtCBP), B. licheniformis DSM13 (BliChi and BliCBP) and Serratia proteamaculans 568 (SpChiB and SpCBP21) were used to study the efficiency and synergistic action of BtChi, BliChi and SpChiB individually with BtCBP, BliCBP or SpCBP21. Chitinase assay revealed that only BtChi and SpChiB showed synergism in hydrolysis of chitin, while there was no increase in products generated by BliChi, in the presence of the three above mentioned CBPs. This suggests that some (specific) CBPs are able to exert a synergistic effect on (specific) chitinases. A mutant of BliChi, designated as BliGH, was constructed by deleting the C-terminal fibronectin III (FnIII) and carbohydrate binding module 5 (CBM5) to assess the contribution of FnIII and CBM5 domains in the synergistic interactions of GH18 chitinases with CBPs. Chitinase assay with BliGH revealed that the accessory domains play a major role in making BliChi an efficient enzyme. We studied binding of BtCBP and BliCBP to α- and ß-chitin. The BtCBP, BliCBP or SpCBP21 did not act synergistically with chitinases in hydrolysis of the chitin, interspersed with other polymers, present in fungal cell walls.

Transglycosylation by chitinase D from Serratia proteamaculans improved through altered substrate interactions.

We describe the improvement of transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced a smaller quantity of TG products for up to 90 min with 2 mm chitotetraose as the substrate and subsequently produced only hydrolytic products. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. The rest of the three mutants, M226A, Y228A, and R284A, displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S, and S116G, at the catalytic groove increased TG activity, whereas W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of Trp-247 at the solvent-accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284A, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared with SpChiD. The double mutant E159D/F64W regained the activity with accumulation of 6.0% chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at the catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of the quantity of TG products produced and the extended duration of TG activity.

Synthesis of long-chain chitooligosaccharides by a hypertransglycosylating processive endochitinase of Serratia proteamaculans 568.

We describe the heterologous expression and characterization of a 407-residue single-domain glycosyl hydrolase family 18 chitinase (SpChiD) from Gram-negative Serratia proteamaculans 568 that has unprecedented catalytic properties. SpChiD was optimally active at pH 6.0 and 40 °C, where it showed a K(m) of 83 mg ml(-1), a k(cat) of 3.9 × 10(2) h(-1), and a k(cat)/K(m) of 4.7 h mg(-1) ml(-1) on colloidal chitin. On chitobiose, the K(m), k(cat), and k(cat)/K(m) were 203 µM, 1.3 × 10(2) h(-1), and 0.62 h(-1) µM(-1), respectively. Hydrolytic activity on chitooligosaccharides (CHOS) and colloidal chitin indicated that SpChiD was an endo-acting processive enzyme, with the unique ability to convert released chitobiose to N-acetylglucosamine, the major end product. SpChiD showed hyper transglycosylation (TG) with trimer-hexamer CHOS substrates, generating considerable amounts of long-chain CHOS. The TG activity of SpChiD was dependent on both the length and concentration of the oligomeric substrate and also on the enzyme concentration. The length and amount of accumulated TG products increased with increases in the length of the substrate and its concentration and decreased with increases in the enzyme concentration. The SpChiD bound to insoluble and soluble chitin substrates despite the absence of accessory domains. Sequence alignments and structural modeling indicated that SpChiD would have a deep substrate-binding groove lined with aromatic residues, which is characteristic of processive enzymes. SpChiD shows a combination of properties that seems rare among family 18 chitinases and that may resemble the properties of human chitotriosidase.

Chitin binding proteins act synergistically with chitinases in Serratia proteamaculans 568.

Genome sequence of Serratia proteamaculans 568 revealed the presence of three family 33 chitin binding proteins (CBPs). The three Sp CBPs (Sp CBP21, Sp CBP28 and Sp CBP50) were heterologously expressed and purified. Sp CBP21 and Sp CBP50 showed binding preference to ß-chitin, while Sp CBP28 did not bind to chitin and cellulose substrates. Both Sp CBP21 and Sp CBP50 were synergistic with four chitinases from S. proteamaculans 568 (Sp ChiA, Sp ChiB, Sp ChiC and Sp ChiD) in degradation of α- and ß-chitin, especially in the presence of external electron donor (reduced glutathione). Sp ChiD benefited most from Sp CBP21 or Sp CBP50 on α-chitin, while Sp ChiB and Sp ChiD had major advantage with these Sp CBPs on ß-chitin. Dose responsive studies indicated that both the Sp CBPs exhibit synergism ≥ 0.2 µM. The addition of both Sp CBP21 and Sp CBP50 in different ratios to a synergistic mixture did not significantly increase the activity. Highly conserved polar residues, important in binding and activity of CBP21 from S. marcescens (Sm CBP21), were present in Sp CBP21 and Sp CBP50, while Sp CBP28 had only one such polar residue. The inability of Sp CBP28 to bind to the test substrates could be attributed to the absence of important polar residues.

Multiple chitinases of an endophytic Serratia proteamaculans 568 generate chitin oligomers.

Serratia proteamaculans 568 genome revealed the presence of four family 18 chitinases (Sp ChiA, Sp ChiB, Sp ChiC, and Sp ChiD). Heterologous expression and characterization of Sp ChiA, Sp ChiB, and Sp ChiC showed that these enzymes were optimally active at pH 6.0-7.0, and 40°C. The three Sp chitinases displayed highest activity/binding to ß-chitin and showed broad range of substrate specificities, and released dimer as major end product from oligomeric and polymeric substrates. Longer incubation was required for hydrolysis of trimer for the three Sp chitinases. The three Sp chitinases released up to tetramers from colloidal chitin substrate. Sp ChiA and Sp ChiB were processive chitinases, while Sp ChiC was a non-processive chitinase. Based on the known structures of ChiA and ChiB from S. marcescens, 3D models of Sp ChiA and Sp ChiB were generated.

Biotechnological approaches to develop bacterial chitinases as a bioshield against fungal diseases of plants.

Fungal diseases of plants continue to contribute to heavy crop losses in spite of the best control efforts of plant pathologists. Breeding for disease-resistant varieties and the application of synthetic chemical fungicides are the most widely accepted approaches in plant disease management. An alternative approach to avoid the undesired effects of chemical control could be biological control using antifungal bacteria that exhibit a direct action against fungal pathogens. Several biocontrol agents, with specific fungal targets, have been registered and released in the commercial market with different fungal pathogens as targets. However, these have not yet achieved their full commercial potential due to the inherent limitations in the use of living organisms, such as relatively short shelf life of the products and inconsistent performance in the field. Different mechanisms of action have been identified in microbial biocontrol of fungal plant diseases including competition for space or nutrients, production of antifungal metabolites, and secretion of hydrolytic enzymes such as chitinases and glucanases. This review focuses on the bacterial chitinases that hydrolyze the chitinous fungal cell wall, which is the most important targeted structural component of fungal pathogens. The application of the hydrolytic enzyme preparations, devoid of live bacteria, could be more efficacious in fungal control strategies. This approach, however, is still in its infancy, due to prohibitive production costs. Here, we critically examine available sources of bacterial chitinases and the approaches to improve enzymatic properties using biotechnological tools. We project that the combination of microbial and recombinant DNA technologies will yield more effective environment-friendly products of bacterial chitinases to control fungal diseases of crops.