Studies on the Hydrolyzing Mechanism for Cyclodextrins of Thermoactinomyces vulgaris R-47 α-Amylase 2 (TVAII). X-Ray Structure of the Mutant E354A Complexed with β-Cyclodextrin, and Kinetic Analyses on Cyclodextrins

Abstract

Crystals of the mutant E354A of Thermoactinomyces vulgaris R-47 α-amylase 2 (TVAII) complexed with β-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 P2₁2₁2₁ and cell dimensions a=111.1 Å, b=117.7 Å, c=113.3 Å, which is almost isomorphous with crystals of the wildtype TVAII, and the structure was refined to an R-factor=0.208 (R_free=0.252) using 3.0 Å resolution data. The refined structure shows that the interactions between Phe286 and two C6 atoms of β-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.