Premium
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
Author(s) -
Abe Akemi,
Yoshida Hiromi,
Tonozuka Takashi,
Sakano Yoshiyuki,
Kamitori Shigehiro
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.05013.x
Subject(s) - pullulan , glycosidic bond , mechanism (biology) , chemistry , amylase , stereochemistry , polysaccharide , biochemistry , enzyme , physics , quantum mechanics
Thermoactinomyces vulgaris R‐47 α‐amylase 1 (TVAI) has unique hydrolyzing activities for pullulan with sequence repeats of α‐(1,4), α‐(1,4), and α‐(1,6) 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‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)] 2 or P5; [Glc‐α‐(1,6)‐Glc‐α‐(1,4)‐Glc‐α‐(1,4)] 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 nonreducing 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.