Crystal Structure of α-Galactosidase from Trichoderma reesei and Its Complex with Galactose: Implications for Catalytic Mechanism

Golubev, A. M.; Nagem, R.A.P.; Brandão-Neto, J.; Neustroev, Kirill N.; Eneyskaya, Elena V.; Kulminskaya, Anna A.; Shabalin, Konstantin A.; Savel'ev, Andrew N.; Polikarpov, Igor

doi:10.1016/j.jmb.2004.03.062

Cited by 70 publications

(43 citation statements)

References 45 publications

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“…Figure 2B is a ''zoomed in'' version of Figure 2A, and shows that this mode has the greatest number and power of &2.0aa phase amplitude densities in the carboxy terminal half of the sequence, the area known to be associated with a greater density of peptide-generated, ''accessible'' CNBr antibody binding locations. This finding is consistent with crystallographic studies indicating that the carboxy end of GAL contains antiparallel -sheets, 79 which we have observed to be characterized by the hydrophobic power spectral mode of &2.0aa. 3 Figure 3 shows the wavelet transformation phase amplitudes of the second eigenfunction, C 2 .…”

Section: Data Relevant To Gal's Hydrophobic Eigenmode Accessibility Asupporting

confidence: 92%

Designing allosteric peptide ligands targeting a globular protein

et al. 2006

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Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein‐targeted, L or D retro‐inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G‐protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, β‐galactosidase, βGAL. Three of the eight 14mer peptides allosterically activated βGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 × 10−9 by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their βGAL binding‐mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the βGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy–entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein‐targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins. © 2006 Wiley Periodicals, Inc. Biopolymers 85: 38–59, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Section: Data Relevant To Gal's Hydrophobic Eigenmode Accessibility Asupporting

confidence: 92%

Designing allosteric peptide ligands targeting a globular protein

et al. 2006

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“…Root mean square deviations between native ScAGal and both complexes for the backbone alignment (all molecule) were 0.2 Å. Previous works (15,28,33) have reported the complex of ␣-galactosidases with the reaction product galactose, and recently, the complexes of human ␣-galactosidase with an intermediate and the substrate melibiose have been reported (9). The main features observed at the galactose-binding pocket (subsite Ϫ1) in the two ScAGal-substrate complexes presented here are conserved with that described previously (for a description of subsites nomenclature within glycosyl hydrolases enzymes see Davies et al (34)).…”

Section: Resultsmentioning

confidence: 68%

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

Fernández-Leiro

Pereira-Rodríguez

Cerdán

et al. 2010

Journal of Biological Chemistry

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␣-Galactosidases catalyze the hydrolysis of terminal ␣-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae ␣-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (␣/␤) 8 barrel and a C-terminal ␤-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the ␤-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.Galactose is present in the oligosaccharides of many plant seeds and is also essential in structures as the hemicelluloses. These polymers build up the plant cell wall and represent a huge storage of carbon within the biosphere and might be an important source of renewable energy (1). In humans, mutations of the ␣-galactosidase gene cause incomplete degradation of glycolipids and glycoproteins, resulting in Fabry disease (2). Different strategies involving recombinant ␣-galactosidases are being developed for the treatment of this disease. Another interesting application is the conversion between the ABO blood groups, determined by differences in polysaccharide structures present in the surface of red blood cells. Some ␣-galactosidases are able to remove the ␣-linked terminal galactose that differs between O antigen (universal blood) and B antigen, and processes involving plant ␣-galactosidases are being developed to obtain O-type blood from B-type donors (3). Furthermore, the activity of ␣-galactosidase is of great interest in many biotechnological applications. It is used to improve the quality and yield of sucrose in the sugar beet industry by achieving an efficient raffinose and other galacto-oligosaccharide hydrolyses. In addition, the processing of soybean-related products and other legume-derived food with this enzyme reduces the content of...

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“…The similarity across the members of the family allowed us to interconvert the ligand recognition through substitution of two residues in the loop. The modularity of the loop is also seen in the structures of other members of the family, including the rice and Hypocrea jecorina ␣-GAL structures, where one turn of helix in the ␤5-␣5 loop is replaced with a shorter loop and a longer ␤5 strand (12,29). In those structures, Cys and Trp residues fill the space of the Glu-203 and Leu-206 residue of ␣-GAL or the Ser-188 and Ala-191 residues in ␣-NAGAL.…”

Section: Discussionmentioning

confidence: 94%

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

Tomašić

Metcalf

Guce

et al. 2010

Journal of Biological Chemistry

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The human lysosomal enzymes ␣-galactosidase (␣-GAL, EC 3.2.1.22) and ␣-N-acetylgalactosaminidase (␣-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of ␣-GAL and ␣-NAGAL. The engineered ␣-GAL (which we call ␣-GAL SA ) retains the antigenicity of ␣-GAL but has acquired the enzymatic specificity of ␣-NAGAL. Conversely, the engineered ␣-NAGAL (which we call ␣-NAGAL EL ) retains the antigenicity of ␣-NAGAL but has acquired the enzymatic specificity of the ␣-GAL enzyme. Comparison of the crystal structures of the designed enzyme ␣-GAL SA to the wild-type enzymes shows that active sites of ␣-GAL SA and ␣-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

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Crystal Structure of α-Galactosidase from Trichoderma reesei and Its Complex with Galactose: Implications for Catalytic Mechanism

Cited by 70 publications

References 45 publications

Designing allosteric peptide ligands targeting a globular protein

Designing allosteric peptide ligands targeting a globular protein

Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases

Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

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