Complexes of Thermoactinomyces vulgaris R-47 α-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with α-(1,6) glycosidic linkages

Abstract

Thermoactinomyces vulgaris. Thermoactinomyces vulgaris R-47 aα-amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α-(l,4), α-(l,4), and α-(l,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes α-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes α-(1,6) glycosidic linkages with a lesser efficiency. X-ray structures of three complexes comprising an inactive mutant TVAI (D356N or D356N/E396Q) and a pullulan model oligosaccharide (P2; [Glc-α-(l,6)-Glc-α-(l,4)-Glc-α-(l,4)] 2 or P5; [GIc-α-(1,6)-Glc-α-(l,4)-Glc-α-(l,4)] 5) were determined. The complex D356N/P2 is a mimic of the enzyme/product complex in the main catalytic reaction of TVAI, and a structural comparison with Aspergillus oryzae α-amylase showed that the (-) subsites of TVAI are responsible for recognizing both starch and pullulan. D356N/E396Q/P2 and D356N/E396Q/P5 provided models of the enzyme/substrate complex recognizing the α-(1,6) glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the non-reducing end of TVAI are effective in the hydrolysis of α-(1,6) glycosidic linkages, leading to weak interactions between substrates and the enzyme. Domain N of TVAI is a starch-binding domain acting as an anchor in the catalytic reaction of the enzyme. In this study, additional substrates were also found to bind to domain N, suggesting that domain N also functions as a pullulan-binding domain. © 2005 The Authors. Journal Compilation © 2005 FEBS.