Abstract
(2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD) and is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation. It catalyzes the oxidation of (2S)-methylsuccinyl-CoA to α,β-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. Here we report the crystal structure of MCD at a resolution of 1.37 Å. The enzyme forms a homodimer of two 60-kDa subunits. Compared with other ACDs, MCD contains an ∼170-residue-long N-terminal extension that structurally mimics a dimer–dimer interface of these enzymes that are canonically organized as tetramers. MCD catalyzes the unprecedented oxidation of an α-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. MCD apparently evolved toward preventing the nonspecific oxidation of succinyl-CoA, which is a close structural homolog of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism. For different metabolic engineering and biotechnological applications, however, an enzyme that can oxidize succinyl-CoA to fumaryl-CoA is sought after. Based on the MCD structure, we were able to shift substrate specificity of MCD toward succinyl-CoA through active-site mutagenesis.
Highlights
Edited by Ruma Banerjee (2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD) and is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation
Other described members of the ACD family are short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD), and very long-chain acyl-CoA dehydrogenase (VLCAD), which are involved in the -oxidation of fatty acids
There, another conserved aromatic residue found in ACDs (PdMCD, Phe-534; VLCAD, Phe-421; MCAD, Tyr-375; and SCAD, Tyr-367) is replaced with an isoleucine (Ile-375 in isobutyryl-CoA dehydrogenase (IBD)), which widens the cavity for substrate accommodation [16]
Summary
PdMCD forms a homodimer, consistent with our gel filtration analysis (Fig. S1). The protein crystallized in the space group P 21 21 21 with one dimer per asymmetric unit. The adenine ring is taking part in cation- stacking interactions between Arg-81 and Trp-476 This interaction might stabilize helix H, which is involved in a crystal contact. The only difference to the central fold of other ACDs is a ϳ10-residue-long loop region connecting -strands 5 and 6 This small loop is flanked by prolines (one N-terminal and three C-terminal) and contains a motif of three aspartates in a row Whereas the C terminus of PdMCD (ϳ390 residues) aligned well with monomeric MCAD, the N terminus is organized in an additional domain. This domain comprises helices AЈ–CЈ, a small intermediate -hairpin motif (strands 1Ј and 2Ј), followed by helices DЈ–EЈ (Fig. 4A).
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