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

Abstract

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.