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

摘要

Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII)can efficiently hydrolyze both starch and cyclomaltooligosaccha-rides (cyclodextrins).The crystal structure of an inactive mutant TVAII in a complex with maltohexaose was determined at a resolution of 2.1 A.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.