Crystal structure of the liganded SCP-2-like domain of human peroxisomal multifunctional enzyme type 2 at 1.75 A resolution.

beta-Oxidation of amino acyl coenzyme A (acyl-CoA) species in mammalian peroxisomes can occur via either multifunctional enzyme type 1 (MFE-1) or type 2 (MFE-2), both of which catalyze the hydration of trans-2-enoyl-CoA and the dehydrogenation of 3-hydroxyacyl-CoA, but with opposite chiral specificity. MFE-2 has a modular organization of three domains. The function of the C-terminal domain of the mammalian MFE-2, which shows similarity with sterol carrier protein type 2 (SCP-2), is unclear. Here, the structure of the SCP-2-like domain comprising amino acid residues 618-736 of human MFE-2 (d Delta h Delta SCP-2L) was solved at 1.75 A resolution in complex with Triton X-100, an analog of a lipid molecule. This is the first reported structure of an MFE-2 domain. The d Delta h Delta SCP-2L has an alpha/beta-fold consisting of five beta-strands and five alpha-helices; the overall architecture resembles the rabbit and human SCP-2 structures. However, the structure of d Delta h Delta SCP-2L shows a hydrophobic tunnel that traverses the protein, which is occupied by an ordered Triton X-100 molecule. The tunnel is large enough to accommodate molecules such as straight-chain and branched-chain fatty acyl-CoAs and bile acid intermediates. Large empty apolar cavities are observed near the exit of the tunnel and between the helices C and D. In addition, the C-terminal peroxisomal targeting signal is ordered in the structure and solvent-exposed, which is not the case with unliganded rabbit SCP-2, supporting the hypothesis of a ligand-assisted targeting mechanism.

Enzymes converting D-3-hydroxyacyl-CoA to trans-2-enoyl-CoA. Microsomal and peroxisomal isoenzymes in rat liver.

Epiermization of 3-hydroxyacyl-CoA, which has been shown to occur as a two-step dehydration-hydration reaction (Hiltunen, J. K., Palosaari, P. M., and Kunau, W.-H. (1989) J. Biol. Chem. 264, 13536-13540; Smeland, E., Jianxun, L., Chu, C., Cuebas, D., and Schulz, H. (1989) Biochem. Biophys. Res. Commun. 160, 988-992) was studied in rat liver. Subcellular fractionations of rat liver on different density gradients revealed a dual distribution of activity, catalyzing dehydration of D-3-hydroxydecanoyl-CoA to trans-2-decenoyl-CoA (hydratase 2) in both peroxisomal and microsomal compartments. Both hydratase 2 activity peaks were separated by dye ligand chromatography from the extract of the combined heavy and light mitochondrial fractions. The activity eluted at low salt was identified as the microsomal isoform and was purified to apparent homogeneity. The M(r) of the native protein (subunit) was found to be 60,000 (31,500), indicating that it is homodimeric. The enzyme activity was inhibited by IgGs isolated from antisera raised against the denatured subunit. The activity eluted at high salt was tentatively identified to be peroxisomal of origin, and the M(r) of the native protein (subunit) was determined to be 62,000 (33,500). The peroxisomal enzyme was not recognized by the antibody to its microsomal counterpart. Analysis of the reaction products of microsomal enzyme activity by gas chromatography-mass spectrometry showed that the enzyme catalyzed reversibly hydration/dehydration between trans-2-enoyl-CoA and D-3-hydroxyacyl-CoA, but L-3-hydroxydecanoyl-CoA was not dehydrated to delta 2-enoyl-CoA compounds. Similar reaction characteristics were also determined for the peroxisomal hydratase by using stereospecific auxiliary enzymes. The present data demonstrate that rat liver contains microsomal and peroxisomal proteins possessing hydratase 2 activities. Although their kinetic properties are similar, immunological data, subunit sizes, and chromatographic evidence clearly indicate that they are different enzymes. Comparisons with other hydratases revealed that the microsomal and peroxisomal hydratase 2 described in the present work are proteins that have not been previously purified.