Crystal Structure of Yellow Meal Worm a-Amylase at 1.64 A Ê Resolution Stefan Strobl 1 , Klaus Maskos 2 , Michael Betz 2 , Georg Wiegand 2 Robert Huber 2 , F. Xavier Gomis-Ru È th 2 * and Rudi Glockshuber 1 * 1 Institut fu È r Molekularbiologie und Biophysik, Eideno Èssische Technische Hochschule Ho Ènggerberg, CH-8093, Zu È rich Switzerland 2 Max-Planck-Institut fu Èr Biochemie, D-82152 Planegg-Martinsried, Germany The three-dimensional structure of the a-amylase from Tenebrio molitor larvae (TMA) has been determined by molecular replacement techniques using diffraction data of a crystal of space group P2 1 2 1 2 1 (a  51.24 A Ê ; b  93.46 A Ê ; c  96.95 A Ê ). The structure has been re®ned to a crystallo- graphic R-factor of 17.7% for 58,219 independent re¯ections in the 7.0 to 1.64 A Ê resolution range, with root-mean-square deviations of 0.008 A Ê for bond lengths and 1.482  for bond angles. The ®nal model comprises all 471 residues of TMA, 261 water molecules, one calcium cation and one chloride anion. The electron density con®rms that the N-terminal gluta- mine residue has undergone a post-transitional modi®cation resulting in a stable 5-oxo-proline residue. The X-ray structure of TMA provides the ®rst three-dimensional model of an insect a-amylase. The monomeric enzyme exhibits an elongated shape approximately 75 A Ê  46 A Ê  40 A Ê and consists of three distinct domains, in line with models for a-amylases from microbial, plant and mammalian origin. However, the structure of TMA re¯ects in the substrate and inhibitor binding region a remarkable difference from mammalian a-amylases: the lack of a highly ¯exible, glycine-rich loop, which has been proposed to be involved in a ``trap-release'' mechanism of substrate hydrolysis by mammalian a-amylases. The structural differences between a-amylases of various origins might explain the speci®city of inhibitors directed exclu- sively against insect a-amylases. # 1998 Academic Press Limited Keywords: a-amylase; X-ray structure; yellow meal worm; Tenebrio molitor; a/b-barrel *Corresponding author Introduction a-Amylases (a-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a family of endo-amylases that catalyze the hydrolysis of a-D-(1,4)-glucan lin- kages in starch components, glycogen and various other related carbohydrates. They play a central role in carbohydrate metabolism of animals, plants, and microorganisms. Moreover, a-amylases have become one of the most valuable enzymes in biotechnology, especially in the food and starch processing industry (Vihinen & Ma È ntsa Èla È , 1989). Many insects, especially those that feed on grain products during larval and/or adult life, depend on their amylases for survival. This is particularly true for the beetle Tenebrio molitor, a cosmopolitan pest of stored products. The catalytic properties of the grub's a-amylase (TMA) have been investi- gated before (Buonocore et al., 1976a; Strobl et al., 1997), as well as its inhibition by several inhibitors in vitro (Applebaum, 1964; Shainkin & Birk, 1970; Silano et al., 1973; Buonocore et al., 1976b; Silano & Zahnley, 1978; Buonocore et al., 1980; Garcõ Âa- Maroto et al., 1991) and in vivo (Applebaum, 1964). In contrast to mammals, the yellow meal worm, i.e. the larva of T. molitor, expresses only a single a-amylase, TMA, consisting of 471 amino acid resi- dues with a molecular mass of 51.3 kDa. We have recently determined the complete amino acid sequence of this monomeric protein by cloning its cDNA (Strobl et al., 1997). TMA is an acidic protein with a calculated pI of 4.3. The pH optimum for Present address: F. X. Gomis-Ru È th, Centre d'Investigacions i Desenvolupament (C.S.I.C.), Carrer Jordi Girona, 18-26, 08034 Barcelona, Spain. Abbreviations used: ANA, Aspergillus niger a-amylase; BAA, barley a-amylase; BLA, Bacillus licheniformis a-amylase; CGT, cyclodextrin glycosyl transferase; PPA, porcine pancreatic a-amylase; r.m.s., root-mean-square; TMA, Tenebrio molitor a-amylase. J. Mol. Biol. (1998) 278, 617±628 0022±2836/98/180617±12 $25.00/0/mb1667 # 1998 Academic Press Limited