Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering

1,3-1,4-beta-Glucanases (or lichenases, EC 3.2.1.73) hydrolyse linear beta-glucans containing beta-1,3 and beta-1,4 linkages such as cereal beta-glucans and lichenan, with a strict cleavage specificity for beta-1,4 glycosidic bonds on 3-O-substituted glucosyl residues. The bacterial enzymes are retaining glycosyl hydrolases of family 16 with a jellyroll beta-sandwich fold and a substrate binding cleft composed of six subsites. The present paper reviews the structure-function aspects of the enzymatic action including mechanistic enzymology, protein engineering and X-ray crystallographic studies.     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.     

Characterization of an exo-beta-1,3-galactanase from Clostridium thermocellum

A gene encoding an exo-beta-1,3-galactanase from Clostridium thermocellum, Ct1,3Gal43A, was isolated. The sequence has similarity with an exo-beta-1,3-galactanase of Phanerochaete chrysosporium (Pc1,3Gal43A). The gene encodes a modular protein consisting of an N-terminal glycoside hydrolase family 43 (GH43) module, a family 13 carbohydrate-binding module (CBM13), and a C-terminal dockerin domain. The gene corresponding to the GH43 module was expressed in Escherichia coli, and the gene product was characterized. The recombinant enzyme shows optimal activity at pH 6.0 and 50 degrees C and catalyzes hydrolysis only of beta-1,3-linked galactosyl oligosaccharides and polysaccharides. High-performance liquid chromatography analysis of the hydrolysis products demonstrated that the enzyme produces galactose from beta-1,3-galactan in an exo-acting manner. When the enzyme acted on arabinogalactan proteins (AGPs), the enzyme produced oligosaccharides together with galactose, suggesting that the enzyme is able to accommodate a beta-1,6-linked galactosyl side chain. The substrate specificity of the enzyme is very similar to that of Pc1,3Gal43A, suggesting that the enzyme is an exo-beta-1,3-galactanase. Affinity gel electrophoresis of the C-terminal CBM13 did not show any affinity for polysaccharides, including beta-1,3-galactan. However, frontal affinity chromatography for the CBM13 indicated that the CBM13 specifically interacts with oligosaccharides containing a beta-1,3-galactobiose, beta-1,4-galactosyl glucose, or beta-1,4-galactosyl N-acetylglucosaminide moiety at the nonreducing end. Interestingly, CBM13 in the C terminus of Ct1,3Gal43A appeared to interfere with the enzyme activity toward beta-1,3-galactan and alpha-l-arabinofuranosidase-treated AGP.     

Molecular cloning, purification and characterization of thermostable beta-1,3-1,4 glucanase from Bacillus subtilis A8-8

A gene encoding a beta-1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant beta-1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60 degrees C, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60 degrees C and retained 30% of its original activity at 70 degrees C for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.     

Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.     

Endo-α-1,4-polygalactosaminidases and their homologs: Structure and evolution

Endo-α-1,4-polygalactosaminidase is a rare enzyme. Its catalytic domain belongs to the GH114 family of glycoside hydrolases. It is shown by phylogenetic analysis that the evolution of the corresponding genes involved duplications, elimination, and horizontal transfer. The domain and secondary structures of endo-α-1,4-polygalactosaminidases are discussed. A hypothesis is put forward as to the structure of the active center of the enzyme. Iterative screening of a protein database reveals evolutionary relationships of the GH114 family with the GH13, GH18, GH20, GH27, GH29, GH31, GH35, GH36, and GH66 families of glycoside hydrolases and with the COG1306, COG1649, COG2342, GHL3, and GHL4 families of proteins with unknown enzymatic functions. Unclassified homologs are grouped into 13 new families of hypothetical glycoside hydrolases: GHL5-GHL15, GH36J, and GH36K.     

Isolation and overexpression of a gene encoding an extracellular beta-(1,3-1,4)-glucanase from Streptococcus bovis JB1.

Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.     

Novel carbohydrate-binding module of beta-1,3-xylanase from a marine bacterium, Alcaligenes sp. strain XY-234

A beta-1,3-xylanase gene (txyA) from a marine bacterium, Alcaligenes sp. strain XY-234, has been cloned and sequenced. txyA consists of a 1,410-bp open reading frame that encodes 469 amino acid residues with a calculated molecular mass of 52,256 Da. The domain structure of the beta-1,3-xylanase (TxyA) consists of a signal peptide of 22 amino acid residues, followed by a catalytic domain which belongs to family 26 of the glycosyl hydrolases, a linker region with one array of DGG and six repeats of DNGG, and a novel carbohydrate-binding module (CBM) at the C terminus. The recombinant TxyA hydrolyzed beta-1,3-xylan but not other polysaccharides such as beta-1,4-xylan, carboxymethylcellulose, curdlan, glucomannan, or beta-1,4-mannan. TxyA was capable of binding specifically to beta-1,3-xylan. The analysis using truncated TxyA lacking either the N- or C-terminal region indicated that the region encoding the CBM was located between residues 376 and 469. Binding studies on the CBM revealed that the K(d) and the maximum amount of protein bound to beta-1,3-xylan were 4.2 microM and 18.2 micromol/g of beta-1,3-xylan, respectively. Furthermore, comparison of the enzymatic properties between proteins with and without the CBM strongly indicated that the CBM of TxyA plays an important role in the hydrolysis of beta-1,3-xylan.     

The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.     

Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance

In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.     

Endo-alpha-1-4-polygalactosaminidases and their homologues: structure and evolution

Endo-alpha-1,4-polygalactosaminidase is a rare enzyme. Its catalytic domain belongs to the GH114 family of glycoside hydrolases. Phylogenetic analysis of the family proteins allowed us to show an important role of duplications, eliminations, and horizontal transfer in the evolution of their genes. Domain structure, the secondary structure, and proposed structure of the active center of the endo-alpha-1,4-polygalactosaminidases are discussed. Evolutionary connections of the GH114 family with GH13, GH18, GH20, GH27, GH29, GH31, GH35, GH36, and GH66 families of glycoside hydrolases, as well as, with COG1306, COG1649, COG2342, GHL3, and GHL4 families of enzymatically uncharacterized proteins have been revealed by iterative screening of the protein database. The unclassified homologues have been grouped into 13 new families of hypothetical glycoside hydrolases: GHL5 - GHL15, GH36J, and GH36K.     

Gene cloning,sequencing, and characterization of a family 9 endoglucanase (CelA) with an unusual pattern of activity from the thermoacidophile Alicyclobacillus acidocaldarius ATCC27009

A gene encoding a beta-1,4-glucanase (CelA) belonging to subfamily E1 of family 9 of glycoside hydrolases was cloned and sequenced from the gram-positive thermoacidophile Alicyclobacillus acidocaldarius strain ATCC27009. The translated protein contains an immunoglobulin-like domain but lacks a cellulose-binding domain. The enzyme, when overproduced in Escherichia coli and purified, displayed a temperature optimum of 70 degrees C and a pH optimum of 5.5. CelA contained one zinc and two calcium atoms. Calcium and zinc are likely to be important for temperature stability. The enzyme was most active against substrates containing beta-1,4-linked glucans (lichenan and carboxy methyl cellulose), but also exhibited activity against oat spelt xylan. A striking pattern of hydrolysis on p-nitrophenyl-glycosides was observed, with highest activity on the cellobioside derivative, some on the cellotetraoside derivative, and none on the glucoside and cellotrioside derivatives. Unmodified cellooligosaccharides were also hydrolyzed by CelA. No signal peptide for transport across the cytoplasmic membrane was detected. This, together with the substrate specificity displayed, near neutral pH optimum and irreversible inactivation at low pH, suggests a role for CelA as a cytoplasmic enzyme for the degradation of imported oligosaccharides.     

An exo-beta-1,3-galactanase having a novel beta-1,3-galactan-binding module from Phanerochaete chrysosporium

An exo-beta-1,3-galactanase gene from Phanerochaete chrysosporium has been cloned, sequenced, and expressed in Pichia pastoris. The complete amino acid sequence of the exo-beta-1,3-galactanase indicated that the enzyme consists of an N-terminal catalytic module with similarity to glycoside hydrolase family 43 and an additional unknown functional domain similar to carbohydrate-binding module family 6 (CBM6) in the C-terminal region. The molecular mass of the recombinant enzyme was estimated as 55 kDa based on SDS-PAGE. The enzyme showed reactivity only toward beta-1,3-linked galactosyl oligosaccharides and polysaccharide as substrates but did not hydrolyze beta-1,4-linked galacto-oligosaccharides, beta-1,6-linked galacto-oligosaccharides, pectic galactan, larch arabinogalactan, arabinan, gum arabic, debranched arabinan, laminarin, soluble birchwood xylan, or soluble oat spelled xylan. The enzyme also did not hydrolyze beta-1,3-galactosyl galactosaminide, beta-1,3-galactosyl glucosaminide, or beta-1,3-galactosyl arabinofuranoside, suggesting that it specifically cleaves the internal beta-1,3-linkage of two galactosyl residues. High performance liquid chromatographic analysis of the hydrolysis products showed that the enzyme produced galactose from beta-1,3-galactan in an exo-acting manner. However, no activity toward p-nitrophenyl beta-galactopyranoside was detected. When incubated with arabinogalactan proteins, the enzyme produced oligosaccharides together with galactose, suggesting that it is able to bypass beta-1,6-linked galactosyl side chains. The C-terminal CBM6 did not show any affinity for known substrates of CBM6 such as xylan, cellulose, and beta-1,3-glucan, although it bound beta-1,3-galactan when analyzed by affinity electrophoresis. Frontal affinity chromatography for the CBM6 moiety using several kinds of terminal galactose-containing oligosaccharides as the analytes clearly indicated that the CBM6 specifically interacted with oligosaccharides containing a beta-1,3-galactobiose moiety. When the degree of polymerization of galactose oligomers was increased, the binding affinity of the CBM6 showed no marked change.     

The three-dimensional structures of two beta-agarases

Agars are important gelifying agents for biochemical use and the food industry. To cleave the beta-1,4-linkages between beta-d-galactose and alpha-l-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called beta-agarases. Beta-agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized. Here we report the first crystal structure of these two beta-agarases. The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 A for beta-agarases A and B, respectively. The structure of beta-agarase A was solved by the multiple anomalous diffraction method, whereas beta-agarase B was solved with molecular replacement using beta-agarase A as model. Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/base residues Glu-152 and Glu-189 for beta-agarases A and B, respectively. The structures of the agarases were compared with those of two lichenases and of a kappa-carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity. The relationship between structure and enzymatic activity of the two beta-agarases from Z. galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides. The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the beta-agarases.     

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.     

Sequence and structural aspects of functional diversification in class I alpha-mannosidase evolution.

MOTIVATION: Class I alpha-mannosidases comprise a homologous and functionally diverse family of glycoside hydrolases. Phylogenetic analysis based on an amino acid sequence alignment of the catalytic domain of class I alpha-mannosidases reveals four well-supported phylogenetic groups within this family. These groups include a number of paralogous members generated by gene duplications that occurred as far back as the initial divergence of the crown-group of eukaryotes. Three of the four phylogenetic groups consist of enzymes that have group-specific biochemical specificity and/or sites of activity. An attempt has been made to uncover the role that natural selection played in the sequence and structural divergence between the phylogenetically and functionally distinct Endoplasmic Reticulum (ER) and Golgi apparatus groups. RESULTS: Comparison of site-specific amino acid variability profiles for the ER and Golgi groups revealed statistically significant evidence for functional diversification at the sequence level and indicated a number of residues that are most likely to have played a role in the functional divergence between the two groups. The majority of these sites appear to contain residues that have been fixed within one organelle-specific group by positive selection. Somewhat surprisingly these selected residues map to the periphery of the alpha-mannosidase catalytic domain tertiary structure. Changes in these peripherally located residues would not seem to have a gross effect on protein function. Thus diversifying selection between the two groups may have acted in a gradual manner consistent with the Darwinian model of natural selection. CONTACT: bishogr@millsaps.edu.     

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35

Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.     

Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: Isolation and characterization of the major form

Five forms of xyloglucan endotransglycosylase/hydrolase (XTH) differing in their isoelectric points (pI) were detected in crude extracts from germinating nasturtium seeds. Without further fractionation, all five forms behaved as typical endotransglycosylases since they exhibited only transglycosylating (XET) activity and no xyloglucan-hydrolysing (XEH) activity. They all were glycoproteins with identical molecular mass, and deglycosylation led to a decrease in molecular mass from approximately 29 to 26.5 kDa. The major enzyme form having pI 6.3, temporarily designated as TmXET(6.3), was isolated and characterized. Molecular and biochemical properties of TmXET(6.3) confirmed its distinction from the XTHs described previously from nasturtium. The enzyme exhibited broad substrate specificity by transferring xyloglucan or hydroxyethylcellulose fragments not only to oligoxyloglucosides and cello-oligosaccharides but also to oligosaccharides derived from β-(1,4)-d-glucuronoxylan, β-(1,6)-d-glucan, mixed-linkage β-(1,3; 1,4)-d-glucan and at a relatively low rate also to β-(1,3)-gluco-oligosaccharides. The transglycosylating activity with xyloglucan as donor and cello-oligosaccharides as acceptors represented 4.6%, with laminarioligosaccharides 0.23%, with mixed-linkage β-(1,3; 1,4)-d-gluco-oligosaccharides 2.06%, with β-(1,4)-d-glucuronoxylo-oligosaccharides 0.31% and with β-(1,6)-d-gluco-oligosaccharides 0.69% of that determined with xyloglucan oligosaccharides as acceptors. Based on the sequence homology of tryptic fragments with the sequences of known XTHs, the TmXET(6.3) was classified into group II of the XTH phylogeny of glycoside hydrolase family GH16.