Phylogenetic analysis of family 6 glycoside hydrolases

Multiple sequence alignment separates members of glycoside hydrolase Family 6 into eight subfamilies: one of mainly actinobacterial endoglucanases (EGs), one of ascomycotal EGs, one of chytridiomycotal EGs and cellobiohydrolases (CBHs), one of actinobacterial and proteobacterial CBHs, one of chytridiomycotal CBHs, two of ascomycotal CBHs, and one of basidiomycotal CBHs. Each also has some proteins of unknown function. Multiple sequence alignment also extends to all of Family 6 the observation that lengths of loops that form the active-site tunnel in CBHs vary among subfamilies, and along with loop conformations, determine enzyme function.     

Hierarchical classification of glycoside hydrolases

This review deals with structural and functional features of glycoside hydrolases, a widespread group of enzymes present in almost all living organisms. Their catalytic domains are grouped into 120 amino acid sequence-based families in the international classification of the carbohydrate-active enzymes (CAZy database). At a higher hierarchical level some of these families are combined in 14 clans. Enzymes of the same clan have common evolutionary origin of their genes and share the most important functional characteristics such as composition of the active center, anomeric configuration of cleaved glycosidic bonds, and molecular mechanism of the catalyzed reaction (either inverting, or retaining). There are now extensive data in the literature concerning the relationship between glycoside hydrolase families belonging to different clans and/or included in none of them, as well as information on phylogenetic protein relationship within particular families. Summarizing these data allows us to propose a multilevel hierarchical classification of glycoside hydrolases and their homologs. It is shown that almost the whole variety of the enzyme catalytic domains can be brought into six main folds, large groups of proteins having the same three-dimensional structure and the supposed common evolutionary origin.     

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.     

Protein structural analysis as a tool for protein design

The analysis of known protein structures is a very valuable and indispensable tool for deciphering the complex rules relating sequence to structure in proteins. On the other hand, the design of novel proteins is certainly the most severe test of our understanding of such rules. In this report we describe our own attempt to develop appropriate tools for the investigation of known protein structure properties and their applications to the design of a novel, all β protein. The success of the design project is a demonstration of the usefulness of careful analysis of the data base of known protein structures. © 1994 John Wiley & Sons, Inc.     

Cellular localization of glycoside hydrolases inBacteroides fragilis

The localizations of six glycosidases produced byBacteroides fragilis—-glucosidase, -glucosidase, -galactosidase, -galactosidase, -N-acetylglucosaminidase, and -l-fucosidase—were studied. Cell fractions and cell extracts were obtained by Triton X-100 release, by disruption by freeze-pressing and sonication, and by osmotic release. Isoelectric focusing of a cytoplasmic and of a Triton X-100 extract of the cell wall fraction was performed and revealed differences in the relative distribution of differently charged forms of -N-acetylglucosaminidase. -Galactosidase and alkaline phosphatase were used as cytoplasmic and periplasmic markers, respectively. It is concluded that inB. fragilis -glucosidase is periplasmic, -l-fucosidase and -galactosidase are cytoplasmic, and -n-acetylglucosaminidase is cell associated and bound to the cell envelope by hydrophobic interactions. -Glucosidase and -galactosidase are localized cytoplasmically and/or located in the cell envelope.     

Purification of glycoside hydrolases from Bacteroides fragilis.

Six glycoside hydrolases in the culture medium of Bacteroides fragilis--alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-L-fucosidase-were systematically purified by ammonium sulfate precipitation, gel filtration chromatography, and density gradient isoelectric focusing. The isoelectric focusing resolved the glycosidases into distinct, well-separated fractions and revealed three differently charged forms of beta-N-acetylglucosaminidase and of alpha-L-fucosidase. Furthermore, alpha-glucosidase and beta-N-acetylglucosaminidase were shown to possess dual affinities for the respective galactoside substrates, and beta-galactosidase also hydrolyzed beta-D-fucoside. alpha-Glucosidase was purified to homogeneity, as indicated by a thin-layer isoelectric focusing zymogram technique. The glycosidases, with exception of beta-glucosidase and the acid alpha-L-fucosidase, were each separated from other glycosidic activities to 99%. The molecular weights varied between 58,000 and 125,000. The pH optima ranged from 4.8 to 6.9.     

Rational design of lipid for membrane protein crystallization

The lipidic cubic phase has been used to grow crystals of membrane proteins for high-resolution structure determination. However, the original, so-called, in meso method does not work reliably at low temperatures, where proteins are generally more stable, because the hosting lipid turns solid. The need existed therefore for a lipid that forms the cubic phase and that supports crystal growth at low temperatures. We created a database of phase diagrams and used it to design such a lipid. X-ray diffraction showed that the new lipid exhibits designed phase behavior. Further, it produces diffraction quality membrane protein crystals by the in meso method at 6 degrees C. This demonstrates that lipidic materials, like their protein counterparts are amenable to rational design. The same approach as used in this study should find application in extending the range of membrane proteins crystallizable by the in meso method and in tailoring transport of cubic phases for controlled delivery and uptake.     

Purification of some glycoside hydrolases by affinity chromatography

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1→3)-linked β-d-glucosyl residues, some of which are attached by (1→6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1→3)-β-d-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an α-d-mannosidase from jack-bean meal.     

Characterization and synergistic interactions of Fibrobacter succinogenes glycoside hydrolases

The objectives of this study were to characterize Fibrobacter succinogenes glycoside hydrolases from different glycoside hydrolase families and to study their synergistic interactions. The gene encoding a major endoglucanase (endoglucanase 1) of F. succinogenes S85 was identified as cel9B from the genome sequence by reference to internal amino acid sequences of the purified native enzyme. Cel9B and two other glucanases from different families, Cel5H and Cel8B, were cloned and overexpressed, and the proteins were purified and characterized. These proteins in conjunction with two predominant cellulases, Cel10A, a chloride-stimulated cellobiosidase, and Cel51A, formerly known as endoglucanase 2 (or CelF), were assayed in various combinations to assess their synergistic interactions using ball-milled cellulose. The degree of synergism ranged from 0.6 to 3.7. The two predominant endoglucanases produced by F. succinogenes, Cel9B and Cel51A, were shown to have a synergistic effect of up to 1.67. Cel10A showed little synergy in combination with Cel9B and Cel51A. Mixtures containing all the enzymes gave a higher degree of synergism than those containing two or three enzymes, which reflected the complementarity in their modes of action as well as substrate specificities.     

Catalytic properties of the bifunctional soybean beta-glucan-binding protein, a member of family 81 glycoside hydrolases

The beta-glucan-binding protein (GBP) of soybean (Glycine max L.) has been shown to contain two different activities. As part of the plasma membrane-localized pathogen receptor complex, it binds a microbial cell wall elicitor, triggering the activation of defence responses. Additionally, the GBP is able to hydrolyze beta-1,3-glucans, as present in the cell walls of potential pathogens. The substrate specificity, the mode of action, and the stereochemistry of the catalysis have been elucidated. This defines for the first time the inverting mode of the catalytic mechanism of glycoside hydrolases belonging to family 81.     

Mapping of genes encoding glycoside hydrolases on the chromosome of Cellulomonas fimi

Cellulomonas fimi genomic DNA was digested with HpaI, MunI, HindIII, and NsiI, producing fragments ranging in size from 20 to 1400 kbp that were resolved by pulsed field gel electrophoresis. Genetic and physical linkages were determined by Southern blotting and were used to construct a genome map. Cellulomonas fimi has a single circular chromosome of approx. 4000 kbp. Except for two closely linked genes, cbh6A and cel5A, the genes known to encode glycoside hydrolases are scattered widely on the chromosome.     

Molecular basis of substrate specificity in family 1 glycoside hydrolases

ss-glycosidases are active upon a large range of substrates. Besides this, subtle changes in the substrate structure may result in large modifications on the ss-glycosidase activity. The characterization of the molecular basis of ss-glycosidases substrate preference may contribute to the comprehension of the enzymatic specificity, a fundamental property of biological systems. ss-glycosidases specificity for the monosaccharide of the substrate nonreducing end (glycone) is controlled by a hydrogen bond network involving at least 5 active site amino acid residues and 4 substrate hydroxyls. From these residues, a glutamate, which interacts with hydroxyls 4 and 6, seems to be a key element in the determination of the preference for fucosides, glucosides and galactosides. Apart from this, interactions with the hydroxyl 2 are essential to the ss-glycosidase activity. The active site residues forming these interactions and the pattern of the hydrogen bond network are conserved among all ss-glycosidases. The region of the ss-glycosidase active site that interacts with the moiety (called aglycone) which is bound to the glycone is formed by several subsites (1 to 3). However, the majority of the non-covalent interactions with the aglycone is concentrated in the first one, which presents a variable spatial structure and amino acid composition. This structural variability is in accordance with the high diversity of aglycones recognized by ss-glycosidases. Hydrophobic interactions and hydrogen bonds are formed with the aglycone, but the manner in which they control the ss-glycosidase specificity still remains to be determined.     

Radiation and functional specialization of the family-3 glycoside hydrolases

A phylogenetic analysis of the glycoside hydrolases of family 3 (GH3s) was conducted in order to infer particular trends in its evolution: functional specialization, gene transfer events, gene duplications and paralogous evolution, and gene deletions. The phylogenetic analysis of GH3s revealed six clusters, i.e., A, B, C, D, E, and F that could fit the definition of 3 sub-families, i.e., AB, AB' and AB". While the sub-families AB' and AB" contain a single cluster, F and E, respectively, the AB sub-family is sub-divided into four clusters. Global analysis of the GH3 phylogenetic tree suggests a primary burst of amplification of the GH3s that might have led to these sub-families. Specializations, gene transfers, and gene duplications among each of these sub-families and phylogenetic clusters might then have occurred and have been inferred. The fine comparison of the enzyme properties and phylogenetic relationships of GH3s allowed to detect common functional groups that belong to the same cluster (D, E or F), or sub-cluster (A1, A2 or B2). The prokaryotic and eukaryotic beta-xylosidases and beta-glucosidases belong to the AB and AB' sub-families, and the N-acetylglucosaminidases are in sub-family AB" (in cluster E). In some instances (B1, B2, C1, C2, and C3), the lack of data and/or the high heterogeneity of the hydrolytic properties did not allow to infer a particular link between an enzyme functional group and a phylogenetic cluster, suggesting the emergence of some highly specialized GH3s.     

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases

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Hydrophobic interaction network analysis for thermostabilization of a mesophilic xylanase

One widely known drawback of enzymes is their instability in diverse conditions. The thermostability of enzymes is particularly relevant for industrial applications because operation at high temperatures has the advantage of a faster reaction rate. Protein stability is mainly determined in this study by intra-molecular hydrophobic interactions that have a collective and 3-dimensional clustering effect. To interpret the thermostability of enzymes, network analysis was introduced into the protein structure, and a network parameter of structural hierarchy, k of k-clique, was used to discern more developed hydrophobic interaction clusters in the protein structure. The favorable clustering conformations of hydrophobic residues, which seemed to be important for protein thermostability, were discovered by the application of a network analysis to hydrophobic interactions of GH11 xylanases. Coordinating higher k-clique hydrophobic interaction clusters through the site-directed mutagenesis of the model enzyme, Bacillus circulans xylanase, stabilized the local structure and thus improved thermostability, such that the enzyme half-life and melting temperature increased by 78 fold and 8.8 °C, respectively. This study highlights the advantages of interpreting collective hydrophobic interaction patterns and their structural hierarchy and the possibility of applying network analysis to the thermostabilization of enzymes.     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.