A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C5N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C5N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C5N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of similar to 10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C5N. C5N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C-56 polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C5N product was observed. This three enzyme pathway is likely the general route to the C5N ring system in other natural products, including the antibiotic moenomycin.