Secondary literature sources for Alpha_L_fucos
The following references were automatically generated.
- Rigden DJ
- Analysis of glycoside hydrolase family 98: catalytic machinery, mechanism and a novel putative carbohydrate binding module.
- FEBS Lett. 2005; 579: 5466-72
- Display abstract
Glycoside hydrolases (GHs) are diverse enzymes of biotechnological and medical importance. Bioinformatics contributes to our understanding of GH structure and function in various ways, including dissection of their typically modular structures and detection of the distant evolutionary relationships between families that often allow for prediction of catalytic sites. Here these twin strands are applied to the recently described GH98 family, the founder member of which is a blood group glycotope-cleaving endo-beta-galactosidase of potential medical importance from Clostridium perfringens. Three domains can be discerned including a central catalytic TIM barrel domain in which putative catalytic residues can be assigned. Distant homologies and domain contexts suggest that the N-terminal domain is a novel carbohydrate binding module.
- Adachi W et al.
- Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17.
- J Mol Biol. 2004; 343: 785-95
- Display abstract
Crystal structures of chitosanase from Bacillus sp. K17 (ChoK) have been determined at 1.5 A resolution in the active form and at 2.0 A resolution in the inactive form. This enzyme belongs to the family GH-8, out of 93 glycoside hydrolase families, and exhibits the substrate specificity of subclass II chitosanase. The catalytic site is constructed on the scaffold of a double-alpha(6)/alpha(6)-barrel, which is formed by six repeating helix-loop-helix motifs. This structure is quite different from those of the GH-46 chitosanases and of GH-5. Structural comparison with CelA (a cellulase belonging to the same family GH-8) suggests that the proton donor Glu122 is conserved, but the proton acceptor is the inserted Glu309 residue, and that the corresponding Asp278 residue in CelA is inactivated in ChoK. The four acidic residues, Asp179, Glu309, Asp183 and Glu107, can be involved in substrate recognition through interactions with the amino groups of the glucosamine residues bound in the -3, -2, -1 and +1 sites, respectively. The hydrophobic Trp235, Trp166, Phe413 and Tyr318 residues are highly conserved for binding of the hexose rings at the -3, -2, +1 and +2 sites, respectively. These structural features indicate that enzymes in GH-8 can be further divided into three subfamilies. Different types of chitosanases are discussed in terms of convergent evolution from different structural ancestors.
- Hidaka M et al.
- Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.
- Structure. 2004; 12: 937-47
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Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.
- Akeboshi H et al.
- Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase.
- Eur J Biochem. 2004; 271: 4420-7
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Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.
- Strohmeier M, Hrmova M, Fischer M, Harvey AJ, Fincher GB, Pleiss J
- Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae.
- Protein Sci. 2004; 13: 3200-13
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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.
- Itoh T, Akao S, Hashimoto W, Mikami B, Murata K
- Crystal structure of unsaturated glucuronyl hydrolase, responsible for the degradation of glycosaminoglycan, from Bacillus sp. GL1 at 1.8 A resolution.
- J Biol Chem. 2004; 279: 31804-12
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Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated d-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 A resolution with a final R-factor of 16.8% for 25 to 1.8 A resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an alpha(6)/alpha(6)-barrel, whose structure is found in the six-hairpin enzyme superfamily of an alpha/alpha-toroidal fold. One side of the UGL alpha(6)/alpha(6)-barrel structure consists of long loops containing three short beta-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp(88) and Asp(149). This was supported by site-directed mutagenesis studies in Asp(88) and Asp(149).
- Pons T, Naumoff DG, Martinez-Fleites C, Hernandez L
- Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68.
- Proteins. 2004; 54: 424-32
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Multiple-sequence alignment of glycoside hydrolase (GH) families 32, 43, 62, and 68 revealed three conserved blocks, each containing an acidic residue at an equivalent position in all the enzymes. A detailed analysis of the site-directed mutations so far performed on invertases (GH32), arabinanases (GH43), and bacterial fructosyltransferases (GH68) indicated a direct implication of the conserved residues Asp/Glu (block I), Asp (block II), and Glu (block III) in substrate binding and hydrolysis. These residues are close in space in the 5-bladed beta-propeller fold determined for Cellvibrio japonicus alpha-L-arabinanase Arb43A [Nurizzo et al., Nat Struct Biol 2002;9:665-668] and Bacillus subtilis endo-1,5-alpha-L-arabinanase. A sequence-structure compatibility search using 3D-PSSM, mGenTHREADER, INBGU, and SAM-T02 programs predicted indistinctly the 5-bladed beta-propeller fold of Arb43A and the 6-bladed beta-propeller fold of sialidase/neuraminidase (GH33, GH34, and GH83) as the most reliable topologies for GH families 32, 62, and 68. We conclude that the identified acidic residues are located at the active site of a beta-propeller architecture in GH32, GH43, GH62, and GH68, operating with a canonical reaction mechanism of either inversion (GH43 and likely GH62) or retention (GH32 and GH68) of the anomeric configuration. Also, we propose that the beta-propeller architecture accommodates distinct binding sites for the acceptor saccharide in glycosyl transfer reaction.
- Miyanaga A, Koseki T, Matsuzawa H, Wakagi T, Shoun H, Fushinobu S
- Crystal structure of a family 54 alpha-L-arabinofuranosidase reveals a novel carbohydrate-binding module that can bind arabinose.
- J Biol Chem. 2004; 279: 44907-14
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As the first known structures of a glycoside hydrolase family 54 (GH54) enzyme, we determined the crystal structures of free and arabinose-complex forms of Aspergillus kawachii IFO4308 alpha-l-arabinofuranosidase (AkAbfB). AkAbfB comprises two domains: a catalytic domain and an arabinose-binding domain (ABD). The catalytic domain has a beta-sandwich fold similar to those of clan-B glycoside hydrolases. ABD has a beta-trefoil fold similar to that of carbohydrate-binding module (CBM) family 13. However, ABD shows a number of characteristics distinctive from those of CBM family 13, suggesting that it could be classified into a new CBM family. In the arabinose-complex structure, one of three arabinofuranose molecules is bound to the catalytic domain through many interactions. Interestingly, a disulfide bond formed between two adjacent cysteine residues recognized the arabinofuranose molecule in the active site. From the location of this arabinofuranose and the results of a mutational study, the nucleophile and acid/base residues were determined to be Glu(221) and Asp(297), respectively. The other two arabinofuranose molecules are bound to ABD. The O-1 atoms of the two arabinofuranose molecules bound at ABD are both pointed toward the solvent, indicating that these sites can both accommodate an arabinofuranose side-chain moiety linked to decorated arabinoxylans.
- Tsai LC, Shyur LF, Lee SH, Lin SS, Yuan HS
- Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes.
- J Mol Biol. 2003; 330: 607-20
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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.
- MacGregor EA, Janecek S, Svensson B
- Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes.
- Biochim Biophys Acta. 2001; 1546: 1-20
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The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.
- Naumoff DG
- beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases.
- Proteins. 2001; 42: 66-76
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Comparison of the amino acid sequences of four families of glycosyl hydrolases reveals that they are homologous and have several common conserved regions. Two of these families contain beta-fructosidases (glycosyl hydrolase families GH32 and GH68) and the other two include alpha-L-arabinases and beta-xylosidases (families GH43 and GH62). The latter two families are proposed to be grouped together with the former two into the beta-fructosidase (furanosidase) superfamily. Several ORFs can be considered as a fifth family of the superfamily on the basis of sequence similarity. It is shown for the first time that a glycosyl hydrolase superfamily can include enzymes with both inversion and retention mechanism of action. Composition of the active center for enzymes of the superfamily is discussed.
- Markovic O, Janecek S
- Pectin degrading glycoside hydrolases of family 28: sequence-structural features, specificities and evolution.
- Protein Eng. 2001; 14: 615-31
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Family 28 belongs to the largest families of glycoside hydrolases. It covers several enzyme specificities of bacterial, fungal, plant and insect origins. This study deals with all available amino acid sequences of family 28 members. First, it focuses on the detailed analysis of 115 sequences of polygalacturonases yielding their evolutionary tree. The large data set allowed modification of some of the existing family 28 sequence characteristics and to draw the sequence features specific for bacterial and fungal exopolygalacturonases discriminating them from the endopolygalacturonases. The evolutionary tree reflects both the taxonomy and specificity so that bacterial, fungal and plant enzymes form their own clusters, the endo- and exo-mode of action being respected, too. The only insect (animal) representative is most related to fungal endopolygalacturonases. The present study brings further: (i) the analysis of available rhamnogalacturonase sequences; (ii) the elucidation of relatedness between the recently added member, the endo-xylogalacturonan hydrolase and the rest of the family; and (iii) revealing the sequence features characteristic of the individual enzyme specificities and the evolutionary relationships within the entire family 28. The disulfides common for the individual enzyme groups were also proposed. With regard to functionally important residues of polygalacturonases, xylogalacturonan hydrolase possesses all of them, while the rhamnogalacturonases, known to lack the histidine residue (His223; Aspergillus niger polygalacturonase II numbering), have a further tyrosine (Tyr291) replaced by a conserved tryptophan. Evolutionarily, the xylogalacturonan hydrolase is most related to fungal exopolygalacturonases and the rhamnogalacturonases form their own cluster on the adjacent branch.
- Harvey AJ, Hrmova M, De Gori R, Varghese JN, Fincher GB
- Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases.
- Proteins. 2000; 41: 257-69
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There are approximately 100 known members of the family 3 group of glycoside hydrolases, most of which are classified as beta-glucosidases and originate from microorganisms. The only family 3 glycoside hydrolase for which a three-dimensional structure is available is a beta-glucan exohydrolase from barley. The structural coordinates of the barley enzyme is used here to model representatives from distinct phylogenetic clusters within the family. The majority of family 3 hydrolases have an NH(2)-terminal (alpha/beta)(8) barrel connected by a short linker to a second domain, which adopts an (alpha/beta)(6) sandwich fold. In two bacterial beta-glucosidases, the order of the domains is reversed. The catalytic nucleophile, equivalent to D285 of the barley beta-glucan exohydrolase, is absolutely conserved across the family. It is located on domain 1, in a shallow site pocket near the interface of the domains. The likely catalytic acid in the barley enzyme, E491, is on domain 2. Although similarly positioned acidic residues are present in closely related members of the family, the equivalent amino acid in more distantly related members is either too far from the active site or absent. In the latter cases, the role of catalytic acid is probably assumed by other acidic amino acids from domain 1.
- Mian IS
- Sequence, structural, functional, and phylogenetic analyses of three glycosidase families.
- Blood Cells Mol Dis. 1998; 24: 83-100
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Glycosidases, which cleave the glycosidic bond between a carbohydrate and another moiety, have been classified into over 63 families. Here, a variety of computational techniques have been employed to examine three families important in normal and abnormal pathology with the aim of developing a framework for future homology modeling, experimental and other studies. Family 1 includes bacterial and archaeal enzymes as well as lactase phlorizin-hydrolase and klotho, glycosidases implicated in disaccharide intolerance II and aging respectively. A statistical model, a hidden Markov model (HMM), for the family 1 glycosidase domain was trained and used as the basis for comparative examination of the conserved and variable sequence and structural features as well as the phylogenetic relationships between family members. Although the structures of four family 1 glycosidases have been determined, this is the first comparative examination of all these enzymes. Aspects that are unique to specific members or subfamilies (substrate binding loops) as well those common to all members (a beta/alpha)8 barrel fold) have been defined. Active site residues in some domains in klotho and lactase-phlorizin hydrolases differ from other members and in one instance may bind but not cleave substrate. The four invariant and most highly conserved residues are not residues implicated in catalysis and/or substrate binding. Of these, a histidine may be involved in transition state stabilization. Glucosylceramidase (family 30) and galactosylceramidase (family 59) are mutated in the lysosomal storage disorders Gaucher disease and Krabbe disease, respectively. HMM-based analysis, structure prediction studies and examination of disease mutations reveal a glycosidase domain common to these two families that also occurs in some bacterial glycosidases. Similarities in the reactions catalyzed by families 30 and 59 are reflected in the presence of a structurally and functionally related (beta/alpha)8 barrel fold related to that in family 1.
- Sanz-Aparicio J, Hermoso JA, Martinez-Ripoll M, Lequerica JL, Polaina J
- Crystal structure of beta-glucosidase A from Bacillus polymyxa: insights into the catalytic activity in family 1 glycosyl hydrolases.
- J Mol Biol. 1998; 275: 491-502
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Family 1 glycosyl hydrolases are a very relevant group of enzymes because of the diversity of biological roles in which they are involved, and their generalized occurrence in all sorts of living organisms. The biological plasticity of these enzymes is a consequence of the variety of beta-glycosidic substrates that they can hydrolyze: disaccharides such as cellobiose and lactose, phosphorylated disaccharides, cyanogenic glycosides, etc. The crystal structure of BglA, a member of the family, has been determined in the native state and complexed with gluconate ligand, at 2.4 A and 2.3 A resolution, respectively. The subunits of the octameric enzyme display the (alpha/beta)8 barrel structural fold previously reported for other family 1 enzymes. However, significant structural differences have been encountered in the loops surrounding the active-center cavity. These differences make a wide and extended cavity in BglA, which seems to be able to accommodate substrates longer than cellobiose, its natural substrate. Furthermore, a third sub-site is encountered, which might have some connection with the transglycosylating activity associated to this enzyme and its certain activity against beta-1,4 oligosaccharides composed of more than two units of glucose. The particular geometry of the cavity which contains the active center of BglA must therefore account for both, hydrolytic and transglycosylating activities. A potent and well known inhibitor of different glycosidases, D-glucono-1,5-lactone, was used in an attempt to define interactions of the substrate with specific protein residues. Although the lactone has transformed into gluconate under crystallizing conditions, the open species still binds the enzyme, the conformation of its chain mimicking the true inhibitor. From the analysis of the enzyme-ligand hydrogen bonding interactions, a detailed picture of the active center can be drawn, for a family 1 enzyme. In this way, Gln20, His121, Tyr296, Glu405 and Trp406 are identified as determinant residues in the recognition of the substrate. In particular, two bidentate hydrogen bonds made by Gln20 and Glu405, could conform the structural explanation for the ability of most members of the family for displaying both, glucosidase and galactosidase activity.
- Burmeister WP, Cottaz S, Driguez H, Iori R, Palmieri S, Henrissat B
- The crystal structures of Sinapis alba myrosinase and a covalent glycosyl-enzyme intermediate provide insights into the substrate recognition and active-site machinery of an S-glycosidase.
- Structure. 1997; 5: 663-75
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BACKGROUND: Myrosinase is the enzyme responsible for the hydrolysis of a variety of plant anionic 1-thio-beta-D-glucosides called glucosinolates. Myrosinase and glucosinolates, which are stored in different tissues of the plant, are mixed during mastication generating toxic by-products that are believed to play a role in the plant defence system. Whilst O-glycosidases are extremely widespread in nature, myrosinase is the only known S-glycosidase. This intriguing enzyme, which shows sequence similarities with O-glycosidases, offers the opportunity to analyze the similarities and differences between enzymes hydrolyzing S- and O-glycosidic bonds. RESULTS: The structures of native myrosinase from white mustard seed (Sinapis alba) and of a stable glycosyl-enzyme intermediate have been solved at 1.6 A resolution. The protein folds into a (beta/alpha)8-barrel structure, very similar to that of the cyanogenic beta-glucosidase from white clover. The enzyme forms a dimer stabilized by a Zn2+ ion and is heavily glycosylated. At one glycosylation site the complete structure of a plant-specific heptasaccharide is observed. The myrosinase structure reveals a hydrophobic pocket, ideally situated for the binding of the hydrophobic sidechain of glucosinolates, and two arginine residues positioned for interaction with the sulphate group of the substrate. With the exception of the replacement of the general acid/base glutamate by a glutamine residue, the catalytic machinery of myrosinase is identical to that of the cyanogenic beta-glucosidase. The structure of the glycosyl-enzyme intermediate shows that the sugar ring is bound via an alpha-glycosidic linkage to Glu409, the catalytic nucleophile of myrosinase. CONCLUSIONS: The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.
- Durand P, Lehn P, Callebaut I, Fabrega S, Henrissat B, Mornon JP
- Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis.
- Glycobiology. 1997; 7: 277-84
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The clan GH-A is a group of more than 200 proteins representing nine established families of glycosyl hydrolases that act on a large variety of substrates. This clan includes five enzymes implicated in lysosomal storage diseases: beta-glucuronidase (Sly disease), beta-glucocerebrosidase (Gaucher disease), beta-galactosidase (Landing disease and Morquito type B disease), beta-mannosidase (mannosidosis) and alpha-L-iduronidase (Hurler-Scheie disease). Examination of known 3D structures from some families of the clan allowed us to deduce structural and functional features shared by these proteins. We then used the hydrophobic cluster analysis method to study the protein sequences of the entire clan. Our results reveal that, despite low levels of sequence identity, all the proteins of the clan (including the aforementioned lysosomal enzymes) likely share a similar catalytic domain consisting of an (alpha/beta)8 barrel with conserved functional amino acids located at the C-terminal ends of six of the eight strands constituting the beta-barrel. Interestingly, several mutations reported to be responsible for lysosomal storage diseases are located within these conserved regions of the lysosomal enzyme catalytic domains.
- Alzari PM, Souchon H, Dominguez R
- The crystal structure of endoglucanase CelA, a family 8 glycosyl hydrolase from Clostridium thermocellum.
- Structure. 1996; 4: 265-75
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BACKGROUND. Cellulases, which catalyze the hydrolysis of glycosidic bonds in cellulose, can be classified into several different protein families. Endoglucanase CelA is a member of glycosyl hydrolase family 8, a family for which no structural information was previously available. RESULTS. The crystal structure of CelA was determined by multiple isomorphous replacement and refined to 1.65 A resolution. The protein folds into a regular (alpha/alpha)6 barrel formed by six inner and six outer alpha helices. Cello-oligosaccharides bind to an acidic cleft containing at least five D-glucosyl-binding subsites (A-E) such that the scissile glycosidic linkage lies between subsites C and D. The strictly conserved residue Glu95, which occupies the center of the substrate-binding cleft and is hydrogen bonded to the glycosidic oxygen, has been assigned the catalytic role of proton donor. CONCLUSIONS. The present analysis provides a basis for modeling homologous family 8 cellulases. The architecture of the active-site cleft, presenting at least five glucosyl-binding subsites, explains why family 8 cellulases cleave cello-oligosaccharide polymers that are at least five D-glycosyl subunits long. Furthermore, the structure of CelA allows comparison with (alpha/alpha)6 barrel glycosidases that are not related in sequence, suggesting a possible, albeit distant, evolutionary relationship between different families of glycosyl hydrolases.
- Bolam DN et al.
- Mannanase A from Pseudomonas fluorescens ssp. cellulosa is a retaining glycosyl hydrolase in which E212 and E320 are the putative catalytic residues.
- Biochemistry. 1996; 35: 16195-204
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Mannanase A (MANA) from Pseudomonas fluorescens, a member of glycosyl hydrolase family 26, was hyperexpressed in Escherichia coli and purified to homogeneity. Analysis of the stereochemical course of mannotetraose hydrolysis by purified MANA showed that the configuration of the anomeric carbon was retained on cleavage of the middle glycosidic bond. These data suggest that the mannanase hydrolyzes mannooligosaccharides by a double-displacement general acid-base mechanism. By hydrophobic cluster analysis (HCA), two glutamate and two aspartate residues were shown to be conserved in all of the glycosyl hydrolase family 26 enzymes analyzed. In addition, HCA suggested that family 26 was related to the GH-A clan (families 1, 2, 5, 10, 30, 35, 39, and 42) of (alpha/beta)8-barrel glycosyl hydrolases, which led to the prediction that E320 and E212 constitute the catalytic nucleophile and acid-base residues, respectively. To investigate the role of these amino acids, site-directed mutagenesis was used to replace the two aspartates with alanine and glutamate, while the two conserved glutamates were changed to alanine and aspartate. The mutant enzymes were purified and their biochemical properties were analyzed. The data showed that neither the D-->A nor the D-->E mutation resulted in a dramatic decrease in enzyme activity, suggesting that the two aspartate residues did not play a pivotal role in catalysis. In contrast, modification of either of the glutamate residues to alanine caused a dramatic decrease in kcat against carob galactomannan, azo-carob galactomannan, mannotetraose and 2,4-dinitrophenyl beta-mannobioside (2,4-DNPM). The E320A mutation did not alter the apparent K(m) (K(m)) of MANA against these substrates, while E212A resulted in a 27-fold decrease in K(m) against 2,4-DNPM. Pre-steady-state kinetics of 2,4-DNPM hydrolysis by E212A showed that there was a rapid burst of 2,4-dinitrophenol release. Circular dichroism and fluorescence spectroscopy indicated that there were no significant differences between the structures of the mutant and wild-type forms of MANA. These data are consistent with E212 and E320 constituting the catalytic acid-base and nucleophile residues of MANA, respectively.
- Dominguez R, Souchon H, Lascombe M, Alzari PM
- The crystal structure of a family 5 endoglucanase mutant in complexed and uncomplexed forms reveals an induced fit activation mechanism.
- J Mol Biol. 1996; 257: 1042-51
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The structures of the Glu140-->Gln mutant of the Clostridium thermocellum endoglucanase CelC in unliganded form (CelC(E140Q)) and in complex with cellohexaose (CelC(E140Q)-Gl(C6)) have been refined to crystallographic R-factors of 19.4% at 1.9 A and 17.8% at 2.3 A resolution, respectively. The structure of CelC(E140Q)-Gl(C6) complex shows two D-glucosyl residues bound to the non-reducing end of the substrate-binding cleft. Comparison of the unliganded and complexes structures reveals conformational changes due to substrate binding, including a significant reorientation of the loop 138-141 which carries the general acid/base catalyst Glu140 in wild-type CelC. Endoglucanase CelC, a family 5 glycohydrolase, exhibits a (beta/alpha)8-fold with an additional subdomain of 54 amino acids inserted between beta-strand 6 and alpha-helix 6. Seven amino acid residues (Arg46, His90, Asn139, Glu140, His198, Tyr200, and Glu280) located close to the catalytic reaction center are strictly conserved in family 5 cellulases. Only three of these residues (His90, Gln140 and Glu280) make direct contacts with the substrate, but all participate in a network of hydrogen bonds which contribute to the stability of the active site architecture and may influence the protonation state of the two catalytic residues. Residue Trp313, which interacts with the nucleophile Glu280 and is within hydrogen bonding distance of the substrate, is involved in a non-proline cis-peptide bond. An aromatic residue occurs at an equivalent position in many other (beta/alpha)8-barrel glycosidases; the presence of a cis-peptide bond at this position in the structures of family 1 beta-glucosidases, family 2 beta-galactosidases, family 5 cellulases, family 17 beta-glucanases, and family 18 chitinases provides further evidence of an evolutionary relationship between glycosyl hydrolases with a (beta/alpha)8- architecture.
- Barrett T, Suresh CG, Tolley SP, Dodson EJ, Hughes MA
- The crystal structure of a cyanogenic beta-glucosidase from white clover, a family 1 glycosyl hydrolase.
- Structure. 1995; 3: 951-60
- Display abstract
BACKGROUND: beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-beta-D-glucosides as well as glucosides with only a carbohydrate moiety (such as cellobiose). The cyanogenic beta-glucosidase from white clover (subsequently referred to as CBG) is responsible for the cleavage of cyanoglucosides. Both CBG and the cyanoglucosides occur within the plant cell wall where they are found in separate compartments and only come into contact when the leaf tissue experiences mechanical damage. This results in the eventual production of hydrogen cyanide which acts as a deterrent to grazing animals. beta-glucosidases have been assigned to particular glycosyl hydrolase families on the basis of sequence similarity; this classification has placed CBG in family 1 (there are a total of over 40 families) for which a three-dimensional structure has so far not been determined. This is the first report of the three-dimensional structure of a glycosyl hydrolase from family 1. RESULTS: The crystal structure of CBG has been determined using multiple isomorphous replacement. The final model has been refined at 2.15 A resolution to an R factor of 18.9%. The overall fold of the molecule is a (beta/alpha)8 [or (alpha/beta)8] barrel (in common with a number of glycosyl hydrolases) with all residues located in a single domain. CONCLUSIONS: Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.
- Henrissat B, Romeu A
- Families, superfamilies and subfamilies of glycosyl hydrolases.
- Biochem J. 1995; 311: 350-1
- Henrissat B, Bairoch A
- New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.
- Biochem J. 1993; 293: 781-8
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301 glycosyl hydrolases and related enzymes corresponding to 39 EC entries of the I.U.B. classification system have been classified into 35 families on the basis of amino-acid-sequence similarities [Henrissat (1991) Biochem. J. 280, 309-316]. Approximately half of the families were found to be monospecific (containing only one EC number), whereas the other half were found to be polyspecific (containing at least two EC numbers). A > 60% increase in sequence data for glycosyl hydrolases (181 additional enzymes or enzyme domains sequences have since become available) allowed us to update the classification not only by the addition of more members to already identified families, but also by the finding of ten new families. On the basis of a comparison of 482 sequences corresponding to 52 EC entries, 45 families, out of which 22 are polyspecific, can now be defined. This classification has been implemented in the SWISS-PROT protein sequence data bank.
- Henrissat B
- A classification of glycosyl hydrolases based on amino acid sequence similarities.
- Biochem J. 1991; 280: 309-16
- Display abstract
The amino acid sequences of 301 glycosyl hydrolases and related enzymes have been compared. A total of 291 sequences corresponding to 39 EC entries could be classified into 35 families. Only ten sequences (less than 5% of the sample) could not be assigned to any family. With the sequences available for this analysis, 18 families were found to be monospecific (containing only one EC number) and 17 were found to be polyspecific (containing at least two EC numbers). Implications on the folding characteristics and mechanism of action of these enzymes and on the evolution of carbohydrate metabolism are discussed. With the steady increase in sequence and structural data, it is suggested that the enzyme classification system should perhaps be revised.