X-ray crystallographic structural studies of α-amylase I fromEisenia fetida

The earthwormEisenia fetidapossesses several cold-active enzymes, including alpha-amylase, beta-glucanase and beta-mannanase.E. fetidapossesses two isoforms of alpha-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10 degrees C. To identify the molecular properties of Ef-Amy I, X-ray crystal structures were determined of the wild type and of the inactive E249Q mutant. Ef-Amy I has structural similarities to mammalian alpha-amylases, including the porcine pancreatic and human pancreatic alpha-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian alpha-amylases indicate that Ef-Amy I has increased structural flexibility and more solvent-exposed acidic residues. These structural features of Ef-Amy I may contribute to its observed catalytic activity at low temperatures, as many cold-adapted enzymes have similar structural properties. The structure of the substrate complex of the inactive mutant of Ef-Amy I shows that a maltohexaose molecule is bound in the active site and a maltotetraose molecule is bound in the cleft between the N- and C-terminal domains. The recognition of substrate molecules by Ef-Amy I exhibits some differences from that observed in structures of human pancreatic alpha-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.