Studies on the hydrolyzing mechanism for cyclodextrins of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII). X-ray structure of the mutant E354A complexed with beta-cyclodextrin, and kinetic analyses on cyclodextrins.
Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) complexed with beta-cyclodextrin were prepared by a soaking method, and the diffraction data were collected at 100 K, using Synchrotron radiation (SPring-8). The crystals belong to an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions a = 111.1 A, b = 117.7 A, c = 113.3 A, which is almost isomorphous with crystals of the wild-type TVAII, and the structure was refined to an R-factor = 0.208 (R(free) = 0.252) using 3.0 A resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of beta-cyclodextrin at the hydrolyzing site are important for TVAII to recognize cyclodextrins as substrates. This observation from the X-ray structure was supported by kinetic analyses of cyclodextrins using the wild-type TVAII, the mutant F286A and F286L. These studies also suggested that the TVAII-hydrolyzing mechanism for cyclodextrins is slightly different from that for starch.
Kondo
S
S
Department of Biotechnology & Life Science, Tokyo University of Agriculture & Technology, Naka-cho, Koganei, Tokyo 184-8588, Japan.
Ohtaki
A
A
Tonozuka
T
T
Sakano
Y
Y
Kamitori
S
S
D019943:Amino Acid Substitution / Q000235:genetics*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D003505:Cyclodextrins; D000516:alpha-Amylases