Clan GH-A Research Articles

Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A.

The three-dimensional structure of Aspergillus aculeatus beta-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (beta/alpha)(8) barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate-enzyme interactions were elucidated by modeling of beta-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A comparison with the substrate binding grooves of other Clan GH-A enzymes suggests that shape complementarity is crucial in determining the specificity of polysaccharidases.

Structural insights into the beta-mannosidase from T. reesei obtained by synchrotron small-angle X-ray solution scattering enhanced by X-ray crystallography.

A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully. The SAXS and crystallographic free atom models exhibit a similar globular form and were used to assess available crystallographic models of glycosyl hydrolases. The structure of the beta-galactosidase, a member of a family 2, clan GHA glycosyl hydrolases, shows an excellent fit to the experimental molecular envelope and distance distribution function of the beta-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of beta-mannosidase quantified by circular dichroism measurements is in a good agreement with that of beta-galactosidase. We show that a comparison of distance distribution functions in combination with 1D and 2D sequence alignment techniques was able to restrict the number of possible structurally homologous proteins. The method could be applied as a general method in structural genomics and related fields once protein solution scattering data are available.

Site actif de la glucocérébrosidase humaine : prédictions structurales et validations expérimentales

Gaucher disease is a lysosomal storage disorder caused by a deficiency in glucocerebrosidase which cleaves the beta-glucosidic linkage of glucosylceramide, a normal intermediate in glycolipid metabolism. Glucocerebrosidase belongs to the clan GH-A of glycoside hydrolases, a large group of enzymes which function with retention of the anomeric configuration at the hydrolysis site. Accurate three-dimensional (3D) structure data for glucocerebrosidase should help to better understand the molecular bases of Gaucher disease. As such 3D structure data were not available, we used the two-dimensional hydrophobic cluster analysis (HCA) method to make structure predictions for the catalytic domains of clan GH-A glycoside hydrolases. We found that all the enzymes of clan GH-A may share a similar catalytic domain consisting of an (alpha/beta)8 barrel with the critical acid/base and nucleophile residues located at the C-terminal ends of strands beta 4 and beta 7, respectively. In the case of glucocerebrosidase, Glu 235 was predicted to be the putative acid/base catalyst whereas the nucleophile was located at Glu 340. Next, in order to obtain experimental evidence supporting these HCA-based predictions, we used retroviral vectors to express, in murine null cells, E235A and E340A mutant proteins, in which alanine residues unable to participate in the enzymatic reaction replace the presumed critical glutamic acid residues. Both mutants were found to be catalytically inactive although they were correctly folded/processed and sorted to the lysosome. Thus, Glu 235 and Glu 340 do indeed play key roles in the active site of human glucocerebrosidase as predicted by the HCA analysis. In a broader perspective, our work points out that bioinformatics approaches may be highly useful for generating structure-function predictions based on sequence-structure interrelationships, especially in the context of a rapid increase in protein sequence information through genome sequencing.

Crystal Structure of Mannanase 26A from Pseudomonas cellulosa and Analysis of Residues Involved in Substrate Binding

The crystal structure of Pseudomonas cellulosa mannanase 26A has been solved by multiple isomorphous replacement and refined at 1.85 A resolution to an R-factor of 0.182 (R-free = 0.211). The enzyme comprises (beta/alpha)(8)-barrel architecture with two catalytic glutamates at the ends of beta-strands 4 and 7 in precisely the same location as the corresponding glutamates in other 4/7-superfamily glycoside hydrolase enzymes (clan GH-A glycoside hydrolases). The family 26 glycoside hydrolases are therefore members of clan GH-A. Functional analyses of mannanase 26A, informed by the crystal structure of the enzyme, provided important insights into the role of residues close to the catalytic glutamates. These data showed that Trp-360 played a critical role in binding substrate at the -1 subsite, whereas Tyr-285 was important to the function of the nucleophile catalyst. His-211 in mannanase 26A does not have the same function as the equivalent asparagine in the other GH-A enzymes. The data also suggest that Trp-217 and Trp-162 are important for the activity of mannanase 26A against mannooligosaccharides but are less important for activity against polysaccharides.

Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis.

Lysosomal storage diseases are due to inherited deficiencies in various enzymes involved in basic metabolic processes. As with other genetic diseases, accurate structure data for these enzymatic proteins should help in better understanding the molecular effects of mutations identified in patients with the corresponding lysosomal diseases; however, no such three-dimensional (3D) structure data are available for many lysosomal enzymes. Thus, we herein intend to illustrate for an audience of molecular geneticists how structure information can nonetheless be obtained via a bioinformatics approach in the case of five human lysosomal glycoside hydrolases. Indeed, using the two-dimensional hydrophobic cluster analysis method to decipher the sequence information available in data banks for the large group of glycoside hydrolases (clan GH-A) to which these human lysosomal enzymes belong, we could deduce structure predictions for their catalytic domains and propose explanations for the molecular effects of mutations described in patients. In addition, in the case of human beta-glucuronidase for which experimental 3D data have been reported, we also show here that bioinformatics methods relying on the available 3D structure information can be used to obtain further insights into the effects of various mutations described in patients with Sly disease. In a broader perspective, our work stresses that, in the context of a rapid increase in protein sequence information through genome sequencing, bioinformatics approaches might be highly useful for generating structure-function predictions based on sequence-structure interrelationships.