Tetracenomycin (Tcm) F2 cyclase, which catalyzes the cyclization of the anthrone Tcm F2 to the naphthacenone Tcm F1 in the biosynthesis of the anthracycline antibiotic Tcm C in Streptomyces glaucescens, has been purified to homogeneity and characterized. The N-terminal sequence of the enzyme establishes that it is encoded by the tcmI gene, whose deduced product has a molecular weight of 12,728. SDS-PAGE analysis gave a single band with a molecular weight of 12,500, whereas gel-filtration chromatography yielded a molecular weight of 37,500, indicating that the Tcm F2 cyclase is a homotrimer in solution. Under pH > or = 8.0, the enzyme catalyzes the cyclization of Tcm F2 to Tcm F1 and has a Km of 121 +/- 18.2 microM and Vmax of 704 +/- 62.3 nmol.min-1.mg-1. In contrast, under pH < or = 6.5, it catalyzes the cyclization of Tcm F2 to 9-decarboxy Tcm F1, a known shunt metabolite of the Tcm C biosynthetic pathway. Tcm F2 cyclase represents the first discrete enzyme for carbon-carbon bond formation via an intramolecular aldol condensation-dehydration mechanism, a key biochemical operation proposed in the early steps of the biosynthesis of all aromatic polyketides.