title: Human Δ³,Δ²-enoyl-CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix-10.
abstract: The catalytic domain of the trimeric human Δ(3),Δ(2)-enoyl-CoA isomerase, type 2 (HsECI2), has the typical crotonase fold. In the active site of this fold two main chain NH groups form an oxyanion hole for binding the thioester oxygen of the 3E- or 3Z-enoyl-CoA substrate molecules. A catalytic glutamate is essential for the proton transfer between the substrate C2 and C4 atoms for forming the product 2E-enoyl-CoA, which is a key intermediate in the β-oxidation pathway. The active site is covered by the C-terminal helix-10. In HsECI2, the isomerase domain is extended at its N terminus by an acyl-CoA binding protein (ACBP) domain. Small angle X-ray scattering analysis of HsECI2 shows that the ACBP domain protrudes out of the central isomerase trimer. X-ray crystallography of the isomerase domain trimer identifies the active site geometry. A tunnel, shaped by loop-2 and extending from the catalytic site to bulk solvent, suggests a likely mode of binding of the fatty acyl chains. Calorimetry data show that the separately expressed ACBP and isomerase domains bind tightly to fatty acyl-CoA molecules. The truncated isomerase variant (without ACBP domain) has significant enoyl-CoA isomerase activity; however, the full-length isomerase is more efficient. Structural enzymological studies of helix-10 variants show the importance of this helix for efficient catalysis. Its hydrophobic side chains, together with residues from loop-2 and loop-4, complete a hydrophobic cluster that covers the active site, thereby fixing the thioester moiety in a mode of binding competent for efficient catalysis.
authors.affiliation: Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland.
authors.forename: Goodluck U
authors.initials: GU
authors.lastname: Onwukwe
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.forename: M Kristian
authors.initials: MK
authors.lastname: Koski
authors.forename: Werner
authors.initials: W
authors.lastname: Schmitz
authors.forename: Rik K
authors.initials: RK
authors.lastname: Wierenga
mesh_terms: D002151:Calorimetry; D002384:Catalysis; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D064006:Dodecenoyl-CoA Isomerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004746:Enoyl-CoA Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D004746:Enoyl-CoA Hydratase; D064006:Dodecenoyl-CoA Isomerase
keywords: ECI; SAXS; crotonase; helix-10; oxyanion hole