Crystal Structure of 2-Enoyl-CoA Hydratase 2 from Human Peroxisomal Multifunctional Enzyme Type 2 M. Kristian Koski, Antti M. Haapalainen, J. Kalervo Hiltunen and Tuomo Glumoff* Department of Biochemistry and Biocenter Oulu, University of Oulu, Box 3000, FIN-90014 Oulu, Finland 2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the b-oxidation of very-long-chain and a-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3 A ˚ resolution. The three- dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I–III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain- length OC8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE- 2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed. q 2004 Elsevier Ltd. All rights reserved. Keywords: b-oxidation; peroxisomes; hot-dog fold; X-ray crystallography; D-bifunctional protein deficiency *Corresponding author Introduction Peroxisomal multifunctional enzyme type 2 (MFE-2, EC 1.1.1.62) is a 79-kDa enzyme composed of three functional units: (3R)-hydroxyacyl-CoA dehydrogenase ((3R)-dehydrogenase), 2-enoyl- CoA hydratase 2 (hydratase 2) and sterol carrier protein 2-like units (SCP-2L) (Figure 1(a)). 1,2 It catalyzes the second and third steps of peroxisomal b-oxidation, and its importance in human lipid metabolism is shown by the severe clinical symp- toms (dysmorphic features, such as macrocephaly and large fontanelles, hypotonia, seizures, etc.) in patients having defects in the gene encoding MFE-2. 3–10 Typical biochemical observations 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Abbreviations used: CoA, coenzyme A; MFE-2, mammalian peroxisomal multifunctional enzyme type 2; Mfe2p, fungal peroxisomal multifunctional enzyme type 2; SCP-2, sterol carrier protein 2; SCP-2L, sterol carrier protein 2-like unit of MFE-2; DHCA, dihydroxycholestanoic acid; THCA, trihydroxycholestanoic acid; SDR, short-chain alcohol dehydrogenase/reductase; CtMfe2p(dh aCb D), 2-enoyl- CoA hydratase 2 unit of C. tropicalis Mfe2p; HsMFE- 2(dDhSCP-2LD), 2-enoyl-CoA hydratase 2 unit of human MFE-2; NCS, non-crystallographic symmetry. E-mail address of the corresponding author: tuomo.glumoff@oulu.fi doi:10.1016/j.jmb.2004.11.009 J. Mol. Biol. (2005) 345, 1157–1169