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.
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 2.1A. 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.
Ohtaki
Akashi
A
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
Mizuno
Masahiro
M
Yoshida
Hiromi
H
Tonozuka
Takashi
T
Sakano
Yoshiyuki
Y
Kamitori
Shigehiro
S
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014364:Tryptophan / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014867:Water / Q000737:chemistry; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D024322:Amino Acids, Aromatic; D009844:Oligosaccharides; D014867:Water; C016549:maltohexaose; D014443:Tyrosine; D014364:Tryptophan; D000516:alpha-Amylases; D000409:Alanine