Structure of a complex of Thermoactinomyces vulgaris R-47 α-amylase 2 with maltohexaose demonstrates the important role of aromatic residues at the reducing end of the substrate binding cleft

Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) can efficiently hydrolyze both starch and cyclomaltooligosaccharides (cyclodextrins). The crystal structure of an inactive mutant TVAII in a complex with maltohexaose was determined at a resolution of 2.1 angstrom. TVAII adopts a dimeric Structure to form two catalytic sites, where substrates are found to bind. At the catalytic site, there are many hydrogen bonds between the enzyme and Substrate at the non-reducing end from the hydrolyzing site, but few hydrogen bonds at the reducing end, where two aromatic residues. Trp356 and Tyr45, make effective interactions with a substrate. Trp356 drastically changes its side-chain conformation to achieve a strong stacking interaction with the substrate, and Tyr45 from another molecule forms a water-mediated hydrogen bond with the substrate. Kinetic analysis of the wild-type and Mutant enzymes in which Trp356 and/or Tyr45 were replaced with Ala suggested that Trp356 and Tyr45 are essential to the catalytic reaction of the enzyme. and that the formation of a dimeric structure is indispensable for TVAII to hydrolyze both starch and cyclodextrins. (c) 2006 Elsevier Ltd. All rights reserved.