pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity?

Glycoside hydrolases of families 32 (GH32) and 68 (GH68) belong to clan GH-J, containing hydrolytic enzymes (sucrose/fructans as donor substrates) and fructosyltransferases (sucrose/fructans as donor and acceptor substrates). In GH32 members, some of the sugar substrates can also function as inhibitors, this regulatory aspect further adding to the complexity in enzyme functionalities within this family. Although 3D structural information becomes increasingly available within this clan and huge progress has been made on structure-function relationships, it is not clear why some sugars bind as inhibitors without being catalyzed. Conserved aspartate and glutamate residues are well known to act as nucleophile and acid/bases within this clan. Based on the available 3D structures of enzymes and enzyme-ligand complexes as well as docking simulations, we calculated the pKa of the acid-base before and after substrate binding. The obtained results strongly suggest that most GH-J members show an acid-base catalyst that is not sufficiently protonated before ligand entrance, while the acid-base can be fully protonated when a substrate, but not an inhibitor, enters the catalytic pocket. This provides a new mechanistic insight aiming at understanding the complex substrate and inhibitor specificities observed within the GH-J clan. Moreover, besides the effect of substrate entrance on its own, we strongly suggest that a highly conserved arginine residue (in the RDP motif) rather than the previously proposed Tyr motif (not conserved) provides the proton to increase the pKa of the acid-base catalyst

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

A molecular envelope of the β-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 Å 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 β-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 β-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of β-mannosidase quantified by circular dichroism measurements is in a good agreement with that of β-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.413093709375Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D., (1989) The Metabolic Basis of Inherited Disease, Vol. II, 6th ed., 2. , McGraw-Hill, Inc., USAWatts, R.W.E., Gibbs, D.A., (1986) Lysosomal Storage Diseases - Biochemical and Clinical Aspects, , Taylor & Francis, Inc., Philadelphia, USARodriguez-Serna, M., Botella-Estrada, R.M.D., Chabas, A., Coll, M., Oliver, V., Febrer, M., Aliaga, A., Angiokeratoma corporis diffusum associated with beta-mannosidase deficiency (1996) Arch. Dermatol., 132 (10), pp. 1219-1222Kuhad, R.C., Singh, A., Eriksson, K.L., Microorganisms and enzymes involved in the degradation of plant fiber cell walls (1997) Biochem. Eng. Biotechnol., 57, pp. 45-125Dey, P.M., Del Campillo, E., Biochemistry of the multiple forms of glycosidases in plants (1984) Adv. Enzymol. Relat. Areas Mol. Biol., 56, pp. 141-249Kulminskaya, A.A., Eneiskaya, E.V., Isaeva-Ivanova, L.S., Savel'ev, A.N., Sidorenko, I.A., Shabalin, K.A., Golubev, A.M., Neustroev, K.N., Enzymatic activity and beta-galactomannan binding property of beta-mannosidase from Trichoderma reesei (1999) Enzyme Microb. Technol., 25 (3-5), pp. 372-377Aparicio, R., Eneiskaya, E.V., Kulminskaya, A.A., Savel'ev, A.N., Golubev, A.M., Neustroev, K.N., Kobarg, J., Polikarpov, I., Crystallization and preliminary X-ray study of beta-mannosidase from Trichoderma reesei (2000) Acta Crystallogr. D, 56, pp. 342-343Shannon, C.E., Weaver, W., (1949) The Mathematical Theory of Communication, , University of Illinois Press, UrbanaMoore, P.B., Small-angle scattering. Information content and error analysis (1980) J. Appl. Crystallogr., 13, pp. 168-175Durand, P., Lehn, P., Callebaut, I., Fabrega, S., Henrissat, B., Mornon, J.P., Active-site motifs of lysosomal acid hydrolases: Invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis (1997) Glycobiology, 7 (2), pp. 277-284Andrade, M.A., Chacon, P., Merelo, J.J., Moran, F., Evaluation of secondary structure of proteins from UV circular dichroism using an unsupervised learning neural network (1993) Protein Eng., 6, pp. 383-390Lobley, A., Whitmore, L., Wallace, B.A., DICHROWEB: An interactive website for the analysis of protein secondary structure from circular dichroism spectra (2002) Bioinformatics, 18, pp. 211-212Mao, D., Wachter, E., Wallace, B.A., Folding of the mitochondrial proton adenosinetriphosphatase proteolipid channel in phospholipid vesicles (1982) Biochemistry, 21 (20), pp. 4960-4968Towns-Andrews, E., Berry, A., Bordas, J., Mant, G.R., Murray, P.K., Roberts, K., Sumner, I., Gabriel, A., Time-resolved X-ray diffraction station: X-ray optics, detectors, and data acquisition (1989) Rev. Sci. Instrum., 60, pp. 2346-2349Lewis, R., Multiwire gas proportional counters: Decrepit antiques or classic performers? (1994) J. Synchrotron Rad., 1, pp. 43-53Boulin, C., Kempf, R., Koch, M.H.J., McLaughlin, S.M., Data appraisal, evaluation and display for synchrotron radiation experiments - Hardware and software (1986) Nucl. Instrum. Methods Phys. Res. A, 249, pp. 399-407Grossmann, J.G., Crawley, J.B., Strange, R., Patel, K.J., Murphy, L.M., Neu, M., Evans, R.W., Hasnain, S.S., The nature of ligand-induced conformational change in transferrin in solution. An investigation using X-ray scattering, XAFS and site-directed mutants (1998) J. Mol. Biol., 279, pp. 461-472Svergun, D.I., Semenyuk, A.V., Feigin, L.A., Small-angle-scattering-data treatment by the regularization method (1988) Acta Crystallogr. A, 44, pp. 244-251Svergun, D.I., Determination of the regularization parameter in indirect-transform methods using perceptual criteria (1992) J. Appl. Crystallogr., 25, pp. 495-503Guinier, A., Fournet, G., (1955) Small-Angle Scattering of X-rays, , John Wiley & Sons, New YorkSvergun, D.I., Petoukhov, M.V., Koch, M.H.J., Determination of Domain Structure of Proteins from X-ray Solution Scattering (2001) Biophys. J., 80, pp. 2946-2953Porod, G., (1982) General Theory in Small-Angle X-ray Scattering, pp. 17-51. , (Glatter, O., and Kratky, O., Eds.), Academic Press, LondonBentley, G.A., Some applications of the phased translation function using calculated phases in molecular replacement (1992) Proceedings of the Daresbury Study Weekend, , DL/SCI/R33Navaza, J., AMoRe: An automated package for molecular replacement (1994) Acta Crystallogr. A, 50, pp. 157-163Hao, Q., Phasing from an envelope (2001) Acta Crystallogr. D, 57, pp. 1410-1414(1994) Acta Crystallogr. D, 50 (4), pp. 760-763Winn, M., Dodson, E.J., Ralph, A., Collaborative computational project, number 4: Providing programs for protein crystallography (1997) Methods Enzymol., 277, pp. 620-633Murshudov, G.N., Vagin, A.A., Dodson, E.J., Refinement of macromolecular structures by the maximum-likelihood method (1997) Acta Crystallogr. D, 53, pp. 240-255Sabini, E., Schubert, H., Murshudov, G., Wilson, K.S., Siika-Aho, M., Penttilä, M., The three-dimensional structure of a Trichoderma reesei beta-mannanase from glycoside hydrolase family 5 (2000) Acta Crystallogr. D, 56, pp. 3-13Kozin, M.B., Svergun, D.I., Automated matching of high- and low-resolution structural models (2001) J. Appl. Crystallogr., 34, pp. 33-41Svergun, D., Barberato, C., Koch, M.H.J., CRYSOL - A program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates (1995) J. Appl. Crystallogr., 28, pp. 768-773Jacobson, R.H., Zhang, X.J., DuBose, R.F., Matthews, B.W., Three-dimensional structure of beta-galactosidase from E. coli (1994) Nature, 369 (6483), pp. 761-766Gaboriaud, C., Bissery, V., Benchetrit, T., Mornon, J.P., Hydrophobic cluster analysis: An efficient new way to compare and analyse amino acid sequences (1987) FEBS Lett., 224 (1), pp. 149-155Lemesle-Varloot, L., Henrissat, B., Gaboriaud, C., Bissery, V., Morgat, A., Mornon, J.P., Hydrophobic cluster analysis: Procedures to derive structural and functional information from 2-D-representation of protein sequences (1990) Biochimie, 72 (8), pp. 555-57

Micelles, Bicelles, Amphipols, Nanodiscs, Liposomes, or Intact Cells: The Hitchhiker’s Guide to the Study of Membrane Proteins by NMR

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