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.
Crystal Structure of the ECH2 Catalytic Domain of CurF from Lyngbya majuscula