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 alpha-glucosidases during the process of terminal starch digestion, and for designing more efficient drugs to control type 2 diabetes or obesity.
Acarbose ; chemistry ; Amino Acid Sequence ; Catalytic Domain ; Crystallography, X-Ray ; Glycoside Hydrolase Inhibitors ; Humans ; Hydrogen Bonding ; Intestines ; enzymology ; Kinetics ; Maltose ; chemistry ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation, Missense ; Oligosaccharides ; chemistry ; Pichia ; Protein Binding ; Recombinant Proteins ; antagonists & inhibitors ; chemistry ; genetics ; Substrate Specificity ; Surface Properties ; alpha-Glucosidases ; chemistry ; genetics

Binding Sites ; Catalytic Domain ; Crystallography, X-Ray ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; N-Acetylgalactosaminyltransferases ; chemistry ; genetics ; metabolism ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Substrate Specificity

Integrase plays a critical role in the recombination of viral DNA into the host genome. Therefore, over the past decade, it has been a hot target of drug design in the fight against type 1 human immunodeficiency virus (HIV-1). Bovine immunodeficiency virus (BIV) integrase has the same function as HIV-1 integrase. We have determined crystal structures of the BIV integrase catalytic core domain (CCD) in two different crystal forms at a resolution of 2.45 Å and 2.2 Å, respectively. In crystal form I, BIV integrase CCD forms a back-to-back dimer, in which the two active sites are on opposite sides. This has also been seen in many of the CCD structures of HIV-1 integrase that were determined previously. However, in crystal form II, BIV integrase CCD forms a novel face-to-face dimer in which the two active sites are close to each other. Strikingly, the distance separating the two active sites is approximately 20 Å, a distance that perfectly matches a 5-base pair interval. Based on these data, we propose a model for the interaction of integrase with its target DNA, which is also supported by many published biochemical data. Our results provide important clues for designing new inhibitors against HIV-1.
Animals ; Catalytic Domain ; genetics ; Cattle ; DNA ; genetics ; DNA, Viral ; HIV-1 ; genetics ; metabolism ; Humans ; Immunodeficiency Virus, Bovine ; enzymology ; genetics ; Integrases ; chemistry ; genetics ; metabolism

Animals ; Centromere ; metabolism ; Chromosomal Proteins, Non-Histone ; metabolism ; Mice ; Models, Molecular ; Synaptonemal Complex ; metabolism