A NOVEL STRATEGY FOR INHIBITION OF ALPHA-AMYLASES - YELLOW MEAL WORM ALPHA-AMYLASE IN COMPLEX WITH THE RAGI BIFUNCTIONAL INHIBITOR AT 2.5 ANGSTROM RESOLUTION

Background: alpha-Amylases catalyze the hydrolysis of alpha-D-(1,4)-gl ucan linkages in starch and related compounds. There is a wide range o f industrial and medical applications for these enzymes and their inhi bitors. The Ragi bifunctional alpha-amylase/trypsin inhibitor (RBI) is the prototype of the cereal inhibitor superfamily and is the only mem ber of this family that inhibits both trypsin and alpha-amylases. The mode of inhibition of alpha-amylases by these cereal inhibitors has so far been unknown. Results: The crystal structure of yellow meal worm alpha-amylase (TMA) in complex with RBI was determined at 2.5 Angstrom resolution, RBI almost completely fills the substrate-binding site of TMA. Specifically, the free N terminus and the first residue (Ser1) o f RBI interact with all three acidic residues of the active site of TM A (Asp185, Glu222 and Asp287), The complex is further stabilized by ex tensive interactions between the enzyme and inhibitor. Although there is no significant structural reorientation in TMA upon inhibitor bindi ng, the N-terminal segment of RBI, which is highly flexible in the fre e inhibitor, adopts a 3(10)-helical conformation in the complex. RBI's trypsin-binding loop is located opposite the alpha-amylase-binding si te, allowing simultaneous binding of alpha-amylase and trypsin, Conclu sions: The binding of RBI to TMA constitutes a new inhibition mechanis m for alpha-amylases and should be general for all alpha-amylase inhib itors of the cereal inhibitor superfamily, Because RBI inhibits two im portant digestive enzymes of animals, it constitutes an efficient plan t defense protein and may be used to protect crop plants from predator y insects.