Crystal Structure of the ECH₂ Catalytic Domain of CurF from Lyngbya majuscula: INSIGHTS INTO A DECARBOXYLASE INVOLVED IN POLYKETIDE CHAIN β-BRANCHING

Curacin A is a mixed polyketide/nonribosomal peptide possessing anti-mitotic and anti-proliferative activity. In the biosynthesis of curacin A, the N-terminal domain of the CurF multifunctional protein catalyzes decarboxylation of 3-methylglutaconyl-acyl carrier protein (ACP) to 3-methylcrotonyl-ACP, the postulated precursor of the cyclopropane ring of curacin A. This decarboxylase is encoded within an "HCS cassette" that is used by several other polyketide biosynthetic systems to generate chemical diversity by introduction of a β-branch functional group to the natural product. The crystal structure of the CurF N-terminal ECH₂ domain establishes that the protein is a crotonase superfamily member. Ala⁷⁸ and Gly¹¹⁸ form an oxyanion hole in the active site that includes only three polar side chains as potential catalytic residues. Site-directed mutagenesis and a biochemical assay established critical functions for His²⁴⁰ and Lys⁸⁶, whereas Tyr⁸² was nonessential. A decarboxylation mechanism is proposed in which His²⁴⁰ serves to stabilize the substrate carboxylate and Lys⁸⁶ donates a proton to C-4 of the acyl-ACP enolate intermediate to form the Δ² unsaturated isopentenoyl-ACP product. The CurF ECH₂ domain showed a 20-fold selectivity for ACP-over CoA-linked substrates. Specificity for ACP-linked substrates has not been reported for any other crotonase superfamily decarboxylase. Tyr⁷³ may select against CoA-linked substrates by blocking a contact of Arg³⁸ with the CoA adenosine 5'-phosphate.