Crystallographic binding studies of rat peroxisomal multifunctional enzyme, type-1 (MFE1) with 3-ketodecanoyl-CoA : capturing active and inactive states of its hydratase and dehydrogenase catalytic sites

Abstract

<div lang="en" class="abs"><p class="abs_header">Abstract</p><p>The peroxisomal multifunctional enzyme type 1 (MFE1) catalyzes two successive reactions in the <em>β</em>-oxidation cycle: the 2<em>E</em>-enoyl-CoA hydratase (ECH) and NAD⁺-dependent 3<em>S</em>-hydroxyacyl-CoA dehydrogenase (HAD) reactions. MFE1 is a monomeric enzyme that has five domains. The N-terminal part (domains A and B) adopts the crotonase fold and the C-terminal part (domains C, D and E) adopts the HAD fold. A new crystal form of MFE1 has captured a conformation in which both active sites are noncompetent. This structure, at 1.7 Å resolution, shows the importance of the interactions between Phe272 in domain B (the linker helix; helix H10 of the crotonase fold) and the beginning of loop 2 (of the crotonase fold) in stabilizing the competent ECH active-site geometry. In addition, protein crystallographic binding studies using optimized crystal-treatment protocols have captured a structure with both the 3-ketodecanoyl-CoA product and NAD⁺ bound in the HAD active site, showing the interactions between 3-ketodecanoyl-CoA and residues of the C, D and E domains. Structural comparisons show the importance of domain movements, in particular of the C domain with respect to the D/E domains and of the A domain with respect to the HAD part. These comparisons suggest that the N-terminal part of the linker helix, which interacts tightly with domains A and E, functions as a hinge region for movement of the A domain with respect to the HAD part.</p></div>