Crystal Structure of Amylomaltase from Thermus aquaticus, a Glycosyltransferase Catalysing the Production of Large Cyclic Glucans Ingo Przylas 1 , Koji Tomoo 1 , Yoshinobu Terada 2 , Takeshi Takaha 2 Kazutoshi Fujii 2 , Wolfram Saenger 1 and Norbert Stra È ter 1 * 1 Institut fu È r Chemie, Abteilung Kristallographie, Freie Universita Èt Berlin, Takustraûe 6, 14195 Berlin, Germany 2 Biochemical Research Laboratory, Ezaki Glico Co. Ltd., 4-6-5 Utajima Nishiyodogawa-ku, Osaka 555- 8502, Japan Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which syn- thesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A Ê resolution. It is a member of the a-amylase superfamily of enzymes, whose core structure consists of a (b, a) 8 barrel. In amylomaltase, the 8-fold symmetry of this barrel is dis- rupted by several insertions between the barrel strands. The largest inser- tions are between the third and ®fth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the a-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the a-amylase superfam- ily, subdomain B2 is unique to amylomaltase. Remarkably, the C-term- inal domain C, which is present in all related enzymes of the a-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the a-amylase super- family, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products. # 2000 Academic Press Keywords: (b, a) 8 barrel; glucanotransferase; disproportionating enzyme; a-amylase family; protein crystallography *Corresponding author Introduction Carbohydrates are essential components of all living organisms and form the most abundant class of biological molecules. Polysaccharides such as starch are an important food reserve in plants and a major nutrient for animals. Whereas higher plants synthesize starch, bacteria, lower eukaryotes and animals accumulate glycogen. Due to the important biological role of these polysaccharides for energy storage and uptake, selective hydrolysis and formation of glycosidic bonds are critical steps for all organisms. Thus, various enzymes have been identi®ed to act on starch. They are classi®ed on the basis of the catalyzed reaction and substrate speci®city (IUBMB, 1992) into glycosyl hydrolases (EC 3.2.1.x) and glycosyl transferases (EC 2.4.x.y). According to further classi®cation based on evol- utionary relationships, sequence homology and common catalytic mechanisms into families and Present address: K. Tomoo, Department of Physical Chemistry, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1064, Japan. Abbreviations used: CA, cycloamylose; CGTase, cyclodextrin glucanotransferase; D-enzyme, disproportionating enzyme; DP, degree of polymerization; MIR, multiple isomorphous replacement; r.m.s.d., root-mean-square-deviation. E-mail address of the corresponding author: strater@chemie.fu-berlin.de doi:10.1006/jmbi.1999.3503 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 296, 873±886 0022-2836/00/030873±14 $35.00/0 # 2000 Academic Press