Human Intestinal Maltase–Glucoamylase: Crystal Structure of the N-Terminal Catalytic Subunit and Basis of Inhibition and Substrate Specificity Lyann Sim 1 , Roberto Quezada-Calvillo 2 , Erwin E. Sterchi 3 , Buford L. Nichols 4 and David R. Rose 1 ⁎ 1 Division of Cancer Genomics and Proteomics, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, 101 College Street, Toronto, ON, Canada M5G 1L7 2 CIEP-Facultad de Ciencias Quimicas, Universidad Autonoma de San Luis Potosí, Av. M. Nava 6, Zona Universitaria, San Luis Potosí, S.L.P. 78360, Mexico 3 Institute of Biochemistry and Molecular Medicine, University of Berne, CH-3012, Switzerland 4 U.S. Department of Agriculture, Agricultural Research Service, Children's Nutrition Research Center and Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030-300, USA Received 2 August 2007; received in revised form 24 October 2007; accepted 26 October 2007 Available online 1 November 2007 Human maltase–glucoamylase (MGAM) is one of the two enzymes responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM is anchored to the small-intestinal brush-border epithelial cells and contains two homologous glycosyl hydrolase family 31 catalytic subunits: an N-terminal subunit (NtMGAM) found near the membrane-bound end and a C-terminal luminal subunit (CtMGAM). In this study, we report the crystal structure of the human NtMGAM subunit in its apo form (to 2.0 Å) and in complex with acarbose (to 1.9 Å). Structural analysis of the NtMGAM–acarbose complex reveals that acarbose is bound to the NtMGAM active site primarily through side-chain interactions with its acarvosine unit, and almost no interactions are made with its glycone rings. These observations, along with results from kinetic studies, suggest that the NtMGAM active site contains two primary sugar subsites and that NtMGAM and CtMGAM differ in their substrate specificities despite their structural relationship. Additional sequence analysis of the CtMGAM subunit suggests several features that could explain the higher affinity of the CtMGAM subunit for longer maltose oligosaccharides. The results provide a structural basis for the complementary roles of these glycosyl hydrolase family 31 subunits in the bioprocessing of complex starch structures into glucose. © 2007 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: maltase–glucoamylase; sucrase–isomaltase; glycosyl hydrolase family 31; crystal structure; acarbose Introduction The human diet relies heavily on plant starches as a source of glucose, a key metabolite in energy metabolism. All starches are composed of two main structural components: amylose, a long linear chain composed of α(1–4)-linked glucosyl units, and amy- lopectin, a larger branched molecule with α(1–4) and α(1–6) linkages. In humans, four enzymes are *Corresponding author. E-mail address: drose@uhnres.utoronto.ca. Abbreviations used: MGAM, maltase–glucoamylase; SI, sucrase–isomaltase; NtMGAM, N-terminal subunit of MGAM; NtSI, N-terminal subunit of SI; CtMGAM, C-terminal subunit of MGAM; CtSI, C-terminal subunit of SI; IP-MGAM, purified full-length MGAM; GH31, glycosyl hydrolase family 31; DMAB, dimethylamine– borane complex; NAG, N-acetylglucosamine; PDB, Protein Data Bank. doi:10.1016/j.jmb.2007.10.069 J. Mol. Biol. (2008) 375, 782–792 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.