A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases.
Park, Y. B. & Cosgrove, D. J. (2012). Plant Physiology, 158(4), 1933-1943.
Xyloglucan is widely believed to function as a tether between cellulose microfibrils in the primary cell wall, limiting cell enlargement by restricting the ability of microfibrils to separate laterally. To test the biomechanical predictions of this “tethered network” model, we assessed the ability of cucumber (Cucumis sativus) hypocotyl walls to undergo creep (long-term, irreversible extension) in response to three family-12 endo-β-1,4-glucanases that can specifically hydrolyze xyloglucan, cellulose, or both. Xyloglucan-specific endoglucanase (XEG from Aspergillus aculeatus) failed to induce cell wall creep, whereas an endoglucanase that hydrolyzes both xyloglucan and cellulose (Cel12A from Hypocrea jecorina) induced a high creep rate. A cellulose-specific endoglucanase (CEG from Aspergillus niger) did not cause cell wall creep, either by itself or in combination with XEG. Tests with additional enzymes, including a family-5 endoglucanase, confirmed the conclusion that to cause creep, endoglucanases must cut both xyloglucan and cellulose. Similar results were obtained with measurements of elastic and plastic compliance. Both XEG and Cel12A hydrolyzed xyloglucan in intact walls, but Cel12A could hydrolyze a minor xyloglucan compartment recalcitrant to XEG digestion. Xyloglucan involvement in these enzyme responses was confirmed by experiments with Arabidopsis (Arabidopsis thaliana) hypocotyls, where Cel12A induced creep in wild-type but not in xyloglucan-deficient (xxt1/xxt2) walls. Our results are incompatible with the common depiction of xyloglucan as a load-bearing tether spanning the 20- to 40-nm spacing between cellulose microfibrils, but they do implicate a minor xyloglucan component in wall mechanics. The structurally important xyloglucan may be located in limited regions of tight contact between microfibrils.
Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.
Anderson, C. T., Carroll, A., Akhmetova, L. & Somerville, C. (2010). Plant Physiology, 152(2), 787-796.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
X4 modules represent a new family of carbohydrate-binding modules that display novel properties.
Bolam, D. N., Xie, H., Pell, G., Hogg, D., Galbraith, G., Henrissat, B. & Gilbert, H. J. (2004). Journal of Biological Chemistry, 279(22), 22953-22963.
The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.
Family 42 carbohydrate-binding modules display multiple arabinoxylan-binding interfaces presenting different ligand affinities.
Ribeiro, T., Santos-Silva, T., Alves, V. D., Dias, F. M. V., Luís, A. S., Prates, J. A. M., Ferraira, L. M. A., Romao, M. J. & Fontes, C. M. G. A. (2010). Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(10), 2054-2062.
Enzymes that degrade plant cell wall polysaccharides display a modular architecture comprising a catalytic domain bound to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs display considerable variation in primary structure and are grouped into 59 sequence-based families organized in the Carbohydrate-Active enZYme (CAZy) database. Here we report the crystal structure of CtCBM42A together with the biochemical characterization of two other members of family 42 CBMs from Clostridium thermocellum. CtCBM42A, CtCBM42B and CtCBM42C bind specifically to the arabinose side-chains of arabinoxylans and arabinan, suggesting that various cellulosomal components are targeted to these regions of the plant cell wall. The structure of CtCBM42A displays a beta-trefoil fold, which comprises 3 sub-domains designated as α, β and γ. Each one of the three sub-domains presents a putative carbohydrate-binding pocket where an aspartate residue located in a central position dominates ligand recognition. Intriguingly, the γ sub-domain of CtCBM42A is pivotal for arabinoxylan binding, while the concerted action of β and γ sub-domains of CtCBM42B and CtCBM42C is apparently required for ligand sequestration. Thus, this work reveals that the binding mechanism of CBM42 members is in contrast with that of homologous CBM13s where recognition of complex polysaccharides results from the cooperative action of three protein sub-domains presenting similar affinities.
Identification of a GH62 α-L-arabinofuranosidase specific for arabinoxylan produced by Penicillium chrysogenum.
Sakamoto, T., Ogura, A., Inui, M., Tokuda, S., Hosokawa, S., Ihara, H. & Kasai, N. (2011). Applied Microbiology and Biotechnology, 90(1), 137-146.
An arabinoxylan arabinofuranohydrolase (AXS5) was purified from the culture filtrate of Penicillium chrysogenum 31B. A cDNA encoding AXS5 (axs5) was isolated by in vitro cloning using the N-terminal amino acid sequence of the native enzyme as a starting point. The deduced amino acid sequence of the axs5 gene has high similarities with those of arabinoxylan arabinofuranohydrolases of Aspergillus niger, Aspergillus tubingensis, and Aspergillus sojae. Module sequence analysis revealed that a “Glyco_hydro_62” was present at position 28–299 of AXS5. This is a family of α-L-arabinofuranosidases which are all members of glycoside hydrolase family 62. Recombinant AXS5 (rAXS5) expressed in Escherichia coli was highly active on arabinoxylan but not on branched sugar beet arabinan. 1H-NMR analysis revealed that the rAXS5 cleaved arabinosyl side-chains linked to C-2 and C-3 of single-substituted xylose residues in arabinoxylan. Semi-quantitative RT-PCR analysis indicated that expression of the axs5 gene in P. chrysogenum 31B was strongly induced by adding D-xylose and arabinoxylan to the culture medium. Moreover, two binding sites of XlnR, a transcriptional activator that regulates the expression of the genes encoding xylanolytic enzymes, are present in the upstream region of the axs5 gene. These results suggest that AXS5 is involved in xylan degradation.
Endo-1,5-α-L-arabinanase from a Subseafloor Bacillus subtilis: Purification, Characterization and Nucleotide Sequence of Its Gene.
Fukada, Y., Koide, O., Miura, T., Kobayashi, T., Inoue, A., & Horikoshi, K. (2011). Journal of applied glycoscience, 58(2), 61-66.
Four arabinan-degrading enzymes are produced by Bacillus subtilis JAM A-3-6, which was isolated from a subseafloor sediment core from 0.5 m below seafloor at a water depth of 1,180 m off the Shimokita Peninsula in Japan. One of the enzymes (AbnAF25) was purified from a culture broth. The molecular mass of the enzyme was around 28 kDa as judged by SDS-polyacrylamide gel electrophoresis. The optimal pH and temperature were pH 6.3 and 60°C in phosphate buffer. AbnAF25 degraded well debranched arabinan, linear arabinan, and arabino-oligosaccharaides, but not arabinoxylan, arabinogalactan or p-nitrophenyl-α-L-arabinofuranoside, which classifies the enzyme as an endo-1,5-α-L-arabinanase. The end products from linear arabinan were mainly arabinose, arabinobiose and arabinotriose. The gene for AbnAF25 was cloned and sequenced. The deduced amino acid sequence of the enzyme revealed the highest similarity to the arabinanase of B. amyloliquefaciens with 83% identity. As AbnAF25 did not show the definite characterization of a subseafloor enzyme, strain JAM A-3-6 seems to be probably dropped or co-sedimented with a soil component.
A novel GH43 α-L-arabinofuranosidase of Penicillium chrysogenum that preferentially degrades single-substituted arabinosyl side chains in arabinan.
Shinozaki, A., Kawakami, T., Hosokawa, S. & Sakamoto, T. (2014). Enzyme and Microbial Technology, 58, 80-86.
We previously described three α-L-arabinofuranosidases (ABFs) secreted by Penicillium chrysogenum 31B. Here, we purified a fourth ABF, termed PcABF43A, from the culture filtrate. The molecular mass of the enzyme was estimated to be 31 kDa. PcABF43A had the highest activity at 35°C and at around pH 5. The enzyme activity was strong on sugar beet L-arabinan but weak on debranched arabinan and arabinoxylan. Low molecular-mass substrates such as p-nitrophenyl α-L-arabinofuranoside, α-1,5-L-arabinooligosaccharides, and branched arabinotriose were highly resistant to the action of PcABF43A. 1H-NMR analysis revealed that PcABF43A hydrolyzed arabinosyl side chains linked to C-2 or C-3 of single-substituted arabinose residues in L-arabinan. Reports concerning enzymes specific for L-arabinan are quite limited. Pcabf43A cDNA encoding PcABF43A was isolated by in vitro cloning. The deduced amino acid sequence of the enzyme shows high similarities with the sequences of other fungal uncharacterized proteins. Semi-quantitative RT-PCR analysis indicated that the Pcabf43A gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was induced with pectic components such as L-arabinose, L-rhamnose, and D-galacturonic acid. Analysis of enzymatic characteristics of PcABF43A, GH51 ABF (AFQ1), and GH54 ABF (AFS1) from P. chrysogenum suggested that PcABF43A and AFS1 function as debranching enzymes and AFQ1 plays a role of saccharification in the degradation of L-arabinan by this fungus.
Nascent pectin formed in Golgi apparatus of pea epicotyls by addition of uronic acids has different properties from nascent pectin at the stage of galactan elongation.
Abdel-Massih, R. M., Rizkallah, H. D., Al-Din, R. S., Baydoun, E. A. H. & Brett, C. T. (2007). Journal of Plant Physiology, 164(1), 1-10.
Microsomal membranes were prepared from etiolated pea (Pisum sativum L.) epicotyls and used to form nascent [Uronic acid-14C]pectin. The enzyme products were characterized by selective enzymic degradation, gel permeation chromatography and analysis of cellulose binding properties. The product obtained had a molecular weight of around 40 kDa, which was significantly lower than that of nascent [Gal-14C]pectin prepared from the same tissues. It is composed mainly of polygalacturonan and perhaps also rhamnogalacturonan (RG-I). Evidence was obtained for the presence of a protein attached to the nascent [Uronic acid-14C]pectin, but it was unaffected by endoglucanase and did not bind to cellulose. Hence, no xyloglucan appeared to be attached to the nascent [Uronic acid-14C]pectin. A model is proposed in which xyloglucan is attached to nascent pectin after formation of homogalacturonan, but before the pectin leaves the Golgi apparatus.
Enzymatic changes in pectic polysaccharides related to the beneficial effect of soaking on bean cooking time.
Martínez‐Manrique, E., Jacinto‐Hernández, C., Garza‐García, R., Campos, A., Moreno, E. & Bernal‐Lugo, I. (2011). Journal of the Science of Food and Agriculture, 91(13), 2394-2398.
Background: Cooking time decreases when beans are soaked first. However, the molecular basis of this decrease remains unclear. To determine the mechanisms involved, changes in both pectic polysaccharides and cell wall enzymes were monitored during soaking. Two cultivars and one breeding line were studied. Results: Soaking increased the activity of the cell wall enzymes rhamnogalacturonase, galactanase and polygalacturonase. Their activity in the cell wall was detected as changes in chemical composition of pectic polysaccharides. Rhamnose content decreased but galactose and uronic acid contents increased in the polysaccharides of soaked beans. A decrease in the average molecular weight of the pectin fraction was induced during soaking. The decrease in rhamnose and the polygalacturonase activity were associated (r = 0.933, P = 0.01, and r = 0.725, P = 0.01, respectively) with shorter cooking time after soaking. Conclusion: Pectic cell wall enzymes are responsible for the changes in rhamnogalacturonan I and polygalacturonan induced during soaking and constitute the biochemical factors that give bean cell walls new polysaccharide arrangements. Rhamnogalacturonan I is dispersed throughout the entire cell wall and interacts with cellulose and hemicellulose fibres, resulting in a higher rate of pectic polysaccharide thermosolubility and, therefore, a shorter cooking time.
Quantification of food polysaccharide mixtures by 1H NMR.
Merkx, D. W., Westphal, Y., van Velzen, E. J., Thakoer, K. V., de Roo, N. & van Duynhoven, J. P. (2017). Carbohydrate Polymers, 179, 379-385.
Polysaccharides are food ingredients that critically determine rheological properties and shelf life. A qualitative and quantitative assessment on food-specific polysaccharide mixtures by 1H NMR is presented. The method is based on the identification of intact polysaccharides, combined with a quantitative analysis of their monosaccharide constituents. Identification of the polysaccharides is achieved by 1H NMR line shape fitting with pure compound spectra. The monomeric composition was determined using the Saeman hydrolysis procedure, followed by direct monosaccharide quantification by 1H NMR. In the quantification, both the monosaccharide degradation during hydrolysis, as well as a correction for the non-instantaneous polysaccharide dissolution were taken into account. These factors were particularly important for the quantification of pectins. The method showed overall good repeatability (RSDr = 4.1 ± 0.9%) and within-laboratory reproducibility (RSDR = 6.1 ± 1.4%) for various food polysaccharides. Polysaccharide mixtures were quantitatively resolved by a non-negative least squares estimation, using identified polysaccharides and their molar monosaccharide stoichiometry as prior knowledge. The accuracy and precision of the presented method make it applicable to a wide range of food polysaccharide mixtures with complex and overlapping 1H NMR spectra.