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Domain swapping in the low-similarity isomerase/hydratase superfamily: The crystal structure of rat mitochondrial ³,²-enoyl-CoA isomerase

¹ Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA
² Department of Chemistry, City College and Graduate School of the City University of New York, New York 10031, USA

(RECEIVED December 21, 2004; FINAL REVISION February 23, 2005; ACCEPTED February 25, 2005)

Two monofunctional ³,²-enoyl-CoA isomerases, one in mitochondria (mECI) and the other in both mitochondria and peroxisomes (pECI), belong to the low-similarity isomerase/hydratase superfamily. Both enzymes catalyze the movement of a double bond from C3 to C2 of an unsaturated acyl-CoA substrate for re-entry into the -oxidation pathway. Mutagenesis has shown that Glu165 of rat mECI is involved in catalysis; however, the putative catalytic residue in yeast pECI, Glu158, is not conserved in mECI. To elucidate whether Glu165 of mECI is correctly positioned for catalysis, the crystal structure of rat mECI has been solved. Crystal packing suggests the enzyme is trimeric, in contrast to other members of the superfamily, which appear crystallographically to be dimers of trimers. The polypeptide fold of mECI, like pECI, belongs to a subset of this superfamily in which the C-terminal domain of a given monomer interacts with its own N-terminal domain. This differs from that of crotonase and 1,4-dihydroxy-2-naphtoyl-CoA synthase, whose C-terminal domains are involved in domain swapping with an adjacent monomer. The structure confirms Glu165 as the putative catalytic acid/base, positioned to abstract the pro-R proton from C2 and reprotonate at C4 of the acyl chain. The large tunnel-shaped active site cavity observed in the mECI structure explains the relative substrate promiscuity in acyl-chain length and stereochemistry. Comparison with the crystal structure of pECI suggests the catalytic residues from both enzymes are spatially conserved but not in their primary structures, providing a powerful reminder of how catalytic residues cannot be determined solely by sequence alignments.

Keywords: domain swapping; low-similarity isomerase/hydratase superfamily; enoyl-CoA isomerase; crystal structure; fatty acid metabolism

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041303705.

Reprint requests to: Jung-Ja P. Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA; e-mail: jjkim{at}mcw.edu; fax: (414) 456-6510.

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