Biologia, Bratislava, 57/Suppl. 11: 71-76, 2002.

ISSN 0006-3088 (Biologia).



Crystal structures and substrate specificities of two a-amylases hydrolyzing cyclodextrins and pullulan from Thermoactinomyces vulgaris R-47.**



Takashi Tonozuka1, Takehiro Yokota1, Kazuhiro Ichikawa1, Masahiro Mizuno1, Shin Kondo2, Atsushi Nishikawa1, Shigehiro Kamitori2 & Yoshiyuki Sakano1*

1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan; tel: ++ 81 42 367 5704, fax: ++ 81 42 367 5705; e-mail:

2 Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan

* corresponding author

Received: October 4, 2001 / Accepted: February 12, 2002



Thermoactinomyces vulgaris R-47 produces two a-amylases, TVA I and TVA II, both of which hydrolyze pullulan to produce panose and also hydrolyze cyclodextrins. Here, we report the crystal structures of a TVA II-substrate complex and of unliganded TVA I. Using a mutated TVA II with modified catalytic residues, we determined the structure of TVA II bound to b-cyclodextrin, methyl b-cyclodextrin, and maltohexaose. TVA II has four domains, A, B, C, and N, and is observed as a dimer in the crystal. The substrates bound to the active-site cleft, which is formed by domains A and B. The overall structures of TVA II and other a-amylase family enzymes are quite different because the similarity of domains other than domains A, B, and C of these enzymes is not found. However, the positions and structures of the sugars bound to TVA II were similar to those observed in the other a-amylase-family enzymes. Crystals of TVA I were obtained using polyethylene glycol 10,000, and the crystal structure of TVA I was determined. Like TVA II, the structure of TVA I was composed of domains A, B, C, and N. However, unlike TVA II, TVA I was observed as a monomeric form in the crystal, and the interaction between domain N and other domains is quite different. When domain N of TVA I and TVA II was deleted, to investigate the roles of domain N for both enzymes, the activities of these mutated enzymes were greatly reduced.


Key words: TVA, Thermoactinomyces vulgaris, pullulan, cyclodextrin, a-amylase, neopullulanase, maltogenic amylase.


** Recent studies show that TVAI and TVAII are not typical “a-amylases”. However, because both enzymes have properties of neopullulanase, cyclomaltodextrinase, and maltogenic amylase, it is difficult to give only one of these specific names for describing TVAI and TVAII. Thus we use the term “a-amylase” in this paper.