X-ray crystallographic structural studies of α-amylase I from Eisenia fetida

Abstract

The earthworm Eisenia fetida possesses several cold-active enzymes, including α-amylase, β-glucanase and β-mannanase. E. fetida possesses two isoforms of α-amylase (Ef-Amy I and II) to digest raw starch. Ef-Amy I retains its catalytic activity at temperatures below 10°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 α-amylases, including the porcine pancreatic and human pancreatic α-amylases. Structural comparisons of the overall structures as well as of the Ca2+-binding sites of Ef-Amy I and the mammalian α-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 α-amylase. This result provides insights into the structural modulation of the recognition of substrates and inhibitors.

Novel cold-adapted raw-starch digesting α-amylases from Eisenia fetida: Gene cloning, expression, and characterization

“…c7174 and c12319. We had previously reported on the cloning and expression of Ef-Amy I in P. pastoris [8] . In this paper, we describe the cloning and expression Ef-Amy II .…”

“…The expression plasmids pPICZαA-Ef-Amy I and pPICZαA-Ef-Amy II were linearized by Sac I and transformed into P. pastoris GS115 cells by electroporation. We previously expressed Ef-Amy I in P. pastoris [8] . Ef-Amy II was expressed using almost the same methods used to express Ef-Amy I.…”

“…The endo-β−1,4-glucanase, mannanase, and α-amylases from E. fetida are cold-adapted enzymes. The crystal structures of Ef-EG2, Ef-Man, and Ef-Amy I were solved [ 4 , 7 , 8 ]. The negatively charged amino acids Asp-and Glu-of Ef-EG2 occupy over two-thirds of the accessible surface area [7] .…”

“…Acidic amino acids on the surface of the protein were correlated with low-temperature adaptation [9] . In a previous paper, we clarified the crystal structural of α-amylase I from E. fetida (Ef-Amy I) [8] . α -Amylases are GHs that degrade α −1,4 glycoside linkages in starch, glycogen, and related α -glucans.…”

“…α -Amylases are GHs that degrade α −1,4 glycoside linkages in starch, glycogen, and related α -glucans. Ef-Amy I has been classified as belonging to the glycoside hydrolase family 13 with a typical ( β/α ) 8 -barrel containing two aspartic acids (D213, D316) and one glutamic acid (E249) that play essential roles in catalysis [8] . Ef-Amy I has structural similarities to mammalian α-amylases, including the porcine pancreatic and human pancreatic α-amylases.…”