Role of Phe286 in the recognition mechanism of cyclomaltooligosaccharides (cyclodextrins) by Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII).X-ray structures of the mutant TVA11s, F286A and F286Y, and kinetic analyses of the Phe286-replaced mutant TVAIIs

Phe286 located in the center of the active site of alpha -amylase 2 from Th ermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substr ate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray s tructures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) an d Tyr (F286Y) were determined at 3.2 Angstrom resolution. Their structures have no significant differences from that of the wild-type enzyme. The kine tic analyses of Phe286-replaced variants showed that the variants with non- aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activi ties than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and th e replacement of Phe286 affects enzymatic activities for CDs more than thos e for starch. (C) 2001 Elsevier Science Ltd. All rights reserved.