Abstract
Human maltase-glucoamylase (MGAM) hydrolyzes linear alpha-1,4-linked oligosaccharide substrates, playing a crucial role in the production of glucose in the human lumen and acting as an efficient drug target for type 2 diabetes and obesity. The amino- and carboxyl-terminal portions of MGAM (MGAM-N and MGAM-C) carry out the same catalytic reaction but have different substrate specificities. In this study, we report crystal structures of MGAM-C alone at a resolution of 3.1 Å, and in complex with its inhibitor acarbose at a resolution of 2.9 Å. Structural studies, combined with biochemical analysis, revealed that a segment of 21 amino acids in the active site of MGAM-C forms additional sugar subsites (+ 2 and + 3 subsites), accounting for the preference for longer substrates of MAGM-C compared with that of MGAM-N. Moreover, we discovered that a single mutation of Trp1251 to tyrosine in MGAM-C imparts a novel catalytic ability to digest branched alpha-1,6-linked oligosaccharides. These results provide important information for understanding the substrate specificity of alphaglucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
Article PDF
Similar content being viewed by others
References
Adams, P.D., Afonine, P.V., Bunkóczi, G., Chen, V.B., Davis, I.W., Echols, N., Headd, J.J., Hung, L.W., Kapral, G.J., Grosse-Kunstleve, R.W., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66, 213–221.
Brayer, G.D., Luo, Y., and Withers, S.G. (1995). The structure of human pancreatic alpha-amylase at 1.8 A resolution and comparisons with related enzymes. Protein Sci 4, 1730–1742.
Brayer, G.D., Sidhu, G., Maurus, R., Rydberg, E.H., Braun, C., Wang, Y., Nguyen, N.T., Overall, C.M., and Withers, S.G. (2000). Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques. Biochemistry 39, 4778–4791.
Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, 905–921.
Cowell, G.M., Tranum-Jensen, J., Sjöström, H., and Norén, O. (1986). Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase. Biochem J 237, 455–461.
Dahlqvist, A., and Telenius, U. (1969). Column chromatography of human small-intestinal maltase, isomaltase and invertase activities. Biochem J 111, 139–146.
Danielsen, E.M. (1994). Dimeric assembly of enterocyte brush border enzymes. Biochemistry 33, 1599–1605.
Emsley, P., and Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132.
Ernst, H.A., Lo Leggio, L., Willemoës, M., Leonard, G., Blum, P., and Larsen, S. (2006). Structure of the Sulfolobus solfataricus alphaglucosidase: implications for domain conservation and substrate recognition in GH31. J Mol Biol 358, 1106–1124.
Gray, G.M., Lally, B.C., and Conklin, K.A. (1979). Action of intestinal sucrase-isomaltase and its free monomers on an alpha-limit dextrin. J Biol Chem 254, 6038–6043.
Heymann, H., Breitmeier, D., and Günther, S. (1995). Human small intestinal sucrase-isomaltase: different binding patterns for maltoand isomaltooligosaccharides. Biol Chem Hoppe Seyler 376, 249–253.
Jenkins, D.J., Taylor, R.H., Goff, D.V., Fielden, H., Misiewicz, J.J., Sarson, D.L., Bloom, S.R., and Alberti, K.G. (1981). Scope and specificity of acarbose in slowing carbohydrate absorption in man. Diabetes 30, 951–954.
Lee, B., and Richards, F.M. (1971). The interpretation of protein structures: estimation of static accessibility. J Mol Biol 55, 379–400.
Low, L.C. (2010). The epidemic of type 2 diabetes mellitus in the Asia-Pacific region. Pediatr Diabetes 11, 212–215.
McCoy, A.J. (2007). Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr D Biol Crystallogr 63, 32–41.
Nichols, B.L., Avery, S., Sen, P., Swallow, D.M., Hahn, D., and Sterchi, E. (2003). The maltase-glucoamylase gene: common ancestry to sucrase-isomaltase with complementary starch digestion activities. Proc Natl Acad Sci U S A 100, 1432–1437.
Nichols, B.L., Eldering, J., Avery, S., Hahn, D., Quaroni, A., and Sterchi, E. (1998). Human small intestinal maltase-glucoamylase cDNA cloning. Homology to sucrase-isomaltase. J Biol Chem 273, 3076–3081.
Nichols, B.L., Quezada-Calvillo, R., Robayo-Torres, C.C., Ao, Z., Hamaker, B.R., Butte, N.F., Marini, J., Jahoor, F., and Sterchi, E.E. (2009). Mucosal maltase-glucoamylase plays a crucial role in starch digestion and prandial glucose homeostasis of mice. J Nutr 139, 684–690.
Otwinowski, Z., and Minor, W. (1997). Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol 276, 307–326.
Qin, X., Ren, L., Yang, X., Bai, F., Wang, L., Geng, P., Bai, G., and Shen, Y. (2011). Structures of human pancreatic α-amylase in complex with acarviostatins: Implications for drug design against type II diabetes. J Struct Biol 1174, 196–202.
Quezada-Calvillo, R., Robayo-Torres, C.C., Opekun, A.R., Sen, P., Ao, Z., Hamaker, B.R., Quaroni, A., Brayer, G.D., Wattler, S., Nehls, M.C., et al. (2007). Contribution of mucosal maltaseglucoamylase activities to mouse small intestinal starch alphaglucogenesis. J Nutr 137, 1725–1733.
Quezada-Calvillo, R., Sim, L., Ao, Z., Hamaker, B.R., Quaroni, A., Brayer, G.D., Sterchi, E.E., Robayo-Torres, C.C., Rose, D.R., and Nichols, B.L. (2008). Luminal starch substrate “brake” on maltaseglucoamylase activity is located within the glucoamylase subunit. J Nutr 138, 685–692.
Rabasa-Lhoret, R., and Chiasson, J.L. (1998). Potential of alphaglucosidase inhibitors in elderly patients with diabetes mellitus and impaired glucose tolerance. Drugs Aging 13, 131–143.
Rossi, E.J., Sim, L., Kuntz, D.A., Hahn, D., Johnston, B.D., Ghavami, A., Szczepina, M.G., Kumar, N.S., Sterchi, E.E., Nichols, B.L., et al. (2006). Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives. FEBS J 273, 2673–2683.
Semenza, G. (1986). Anchoring and biosynthesis of stalked brush border membrane proteins: glycosidases and peptidases of enterocytes and renal tubuli. Annu Rev Cell Biol 2, 255–313.
Sim, L., Quezada-Calvillo, R., Sterchi, E.E., Nichols, B.L., and Rose, D.R. (2008). Human intestinal maltase-glucoamylase: crystal structure of the N-terminal catalytic subunit and basis of inhibition and substrate specificity. J Mol Biol 375, 782–792.
Sim, L., Willemsma, C., Mohan, S., Naim, H.Y., Pinto, B.M., and Rose, D.R. (2010). Structural basis for substrate selectivity in human maltase-glucoamylase and sucrase-isomaltase N-terminal domains. J Biol Chem 285, 17763–17770.
Van Beers, E.H., Büller, H.A., Grand, R.J., Einerhand, A.W., and Dekker, J. (1995). Intestinal brush border glycohydrolases: structure, function, and development. Crit Rev Biochem Mol Biol 30, 197–262.
Author information
Authors and Affiliations
Corresponding authors
Additional information
These authors contributed equally to the work.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Ren, L., Qin, X., Cao, X. et al. Structural insight into substrate specificity of human intestinal maltase-glucoamylase. Protein Cell 2, 827–836 (2011). https://doi.org/10.1007/s13238-011-1105-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13238-011-1105-3