Domain swapping in the low‐similarity isomerase/hydratase superfamily: The crystal structure of rat mitochondrial Δ³, Δ²‐enoyl‐CoA isomerase

Abstract

Two monofunctional D 3 ,D 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 b-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 To metabolize unsaturated fatty acids, eukaryotes require two auxiliary enzymes, 2,4-dienoyl-CoA reductase (DCR) and D 3,D 2 -enoyl-CoA isomerase (ECI; EC 5.3.3.8). ECI catalyzes the movement of the double bond between carbons 3 and 4 of the acyl chain to carbons 2 and 3, allowing the acyl-CoA to re-enter the b-oxidation pathway at the second step. ECI differs from DCR in that it is essential for the metabolism of all fatty acids with double bonds at odd as well as even carbon positions (Schulz and Kunau 1987). However, like DCR, ECI is relatively promiscuous, catalyzing the isomerization of both cis and trans unsaturated fatty acyl chains (Stoffel et al. 1964;Palosaari et al. 1990;Zhang et al. 2002) (Palosaari et al. 1990;Zhang et al. 2002), as well as in the peroxisomes of yeast (Geisbrecht et al. 1998;Gurvitz et al. 1998). Sequence analysis shows they belong to the low-similarity isomerase/hydratase superfamily (LSI/H) (Muller-Newen et al. 1995;Holden et al. 2001), with the mammalian forms grouped into four classes based on their substrate specificity, organelle location, and physical composition: mitochondrial enoyl-CoA isomerase (mECI), mitochondrial enoyl-CoA hydratase 1 (crotonase), peroxisom...

Human Δ3,Δ2‐enoyl‐CoA isomerase, type 2: a structural enzymology study on the catalytic role of its ACBP domain and helix‐10

“…3B) the catalytic base Glu136 protrudes into the catalytic site out of a different position of loop-4 ( Fig. 3) [8,9]. This position is the same as that of the catalytic glutamate Glu164 in the ECH structure ( Fig.…”

“…Rodents possess a third mitochondrial ECI, which has been identified recently and which is homologous to ECI2 . Structures have been described for ECI1 from rat mitochondria (1XX4) and human mitochondria (1SG4) , MFE1 from rat peroxisomes (2X58) and ECI from yeast peroxisomes (1PJH) . Here, we report on structural enzymological studies of human ECI2 (HsECI2).…”

Mechanisms and structures of crotonase superfamily enzymes – How nature controls enolate and oxyanion reactivity

“…In support of the idea of a common platform on which activities can be relatively easily evolved or interchanged, there is experimental evidence that some of the reaction types catalyzed by CS members can be interchanged, e.g., mutation of 4-CBD (V) to introduce active site residues present in ECH (XI) results in a 4-CBD (V) mutant with previously absent hydratase activity [26] (see CoA-dependent CS members and their mechanisms below). Structures of all CS members crystallized in the presence of a CoA substrate (or analogue) show the active sites to be spatially similar with respect to binding of the CoA portion of the substrate [19]. The CoA portion of CS substrates is usually observed to be bound in the extended binding pocket in a Ushape, centered around the pyrophosphate group (Fig.…”

“…Domain swapping is proposed as a mechanism by which CS monomers may have evolved to form intertwined oligomers [18]. Members of the CS have been classified into three groups according to the relationship of the Cterminal a-helical domain of individual subunits with respect to the rest of the structure into those that undergo: (i) monomer self-association, (ii) intratrimer associations or (iii) inter-trimer associations [19,20]. In the monomer self-association fold, the Cterminal domain folds back over the core to cover the active site of its own monomer, for example as in…”

“…Menaquinone biosynthesis is therefore a potential target for drugs to treat tuberculosis [20]. Crystallographic analyses reveal that MenB (IX) is unusual as it is the only CS non-carboxyltransferase whose C-terminal domain crosses the trimer-trimer interface [19,20] (see Biotin-independent CS decarboxylases below for CS carboxyltransferases). To date, the MenB (IX) reaction is a rare example of intramolecular C-C bond formation within the CS (along with CarB, XVI), although other members catalyze C-C fragmentation.…”