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Thermoactinomyces vulgarisR-47a-amylase 1 (TVAI) has unique hydrolyz-ing activities for pullulan with sequence repeats of a-(1,4), a-(1,4), and a-(1,6) glycosidic linkages, as well as for starch. TVAI mainly hydrolyzes a-(1,4) glycosidic linkages to produce a panose, but it also hydrolyzes a-(1,6) glycosidic linkages with a lesser efficiency. | ềFEBS Journal Complexes of Thermoactinomyces vulgaris R-47 a-amylase 1 and pullulan model oligossacharides provide new insight into the mechanism for recognizing substrates with a- 1 6 glycosidic linkages Akemi Abe1 Hiromi Yoshida1 2 Takashi Tonozuka3 Yoshiyuki Sakano3 and Shigehiro Kamitori1 2 1 Graduate Schoolof Medicine Kagawa University Japan 2 Molecular Structure Research Group Information Technology Center Kagawa University Japan 3 Department of Applied BiologicalScience Tokyo University of Agriculture Technology Japan Keywords X-ray structure a-amylase pullulan enzymatic glucoside hydrolysis Thermoactinomyces vulgaris Correspondence S. Kamitori Molecular Structure Research Group Information Technology Center Kagawa University 1750-1 Ikenobe Miki-cho Kita-gun kagawa 761-0793 Japan Tel Fax 81 87 891 2421 E-mail kamitori@med.kagawa-u.ac.jp Received 16 August 2005 revised 4 October 2005 accepted 11 October 2005 doi 10.1111 j.1742-4658.2005.05013.x Thermoactinomyces vulgaris R-47 a-amylase 1 TVAI has unique hydrolyzing activities for pullulan with sequence repeats of a- 1 4 a- 1 4 and a- 1 6 glycosidic linkages as well as for starch. TVAI mainly hydrolyzes a- 1 4 glycosidic linkages to produce a panose but it also hydrolyzes a- 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-a- 1 6 -Glc-a- 1 4 -Glc-a- 1 4 2 or P5 Glc-a- 1 6 -Glc-a- 1 4 -Glc-a- 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 a-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 a- 1 6 glycosidic linkage at the hydrolyzing site. They showed that only subsites -1 and -2 at the nonreducing end .
