The prodiginines are a group of specialized metabolites that share a 4-methoxypyrrolyldipyrromethene core structure. Streptorubin B is a structurally remarkable member of the prodiginine group produced by Streptomyces coelicolor A3(2) and other actinobacteria. It is biosynthesized from undecylprodigiosin by an oxidative carbocyclization catalyzed by the Rieske oxygenase-like enzyme RedG. Undecylprodigiosin derives from the RedH-catalyzed condensation of 2-undecylpyrrole and 4-methoxy-2, 2′-bipyrrole-5-carboxaldehyde (MBC). To probe the mechanism of the RedG-catalyzed reaction, we synthesized 2-(5-pentoxypentyl)-pyrrole, an analogue of 2-undecylpyrrole with an oxygen atom next to the site of C-C bond formation, and fed it, along with synthetic MBC, to Streptomyces albus expressing redH and redG. This resulted in the production of the 6′-oxa analogue of undecylprodigiosin. In addition, a small amount of a derivative of this analogue lacking the n-pentyl group was produced, consistent with a RedG catalytic mechanism involving hydrogen abstraction from the alkyl chain of undecylprodigiosin prior to pyrrole functionalization. To investigate the stereochemistry of the RedG-catalyzed oxidative carbocyclization, [7′-2H](7′R)-2-undecylpyrrole and [7′-2H](7′S)-2-undecylpyrrole were synthesized and fed separately, along with MBC, to S. albus expressing redH and redG. Analysis of the extent of deuterium incorporation into the streptorubin B produced in these experiments showed that the pro-R hydrogen atom is abstracted from C-7′ of undecylprodigiosin and that the reaction proceeds with inversion of configuration at C-7′. This contrasts sharply with oxidative heterocyclization reactions catalyzed by other nonheme iron-dependent oxygenase-like enzymes, such as isopenicillin N synthase and clavaminate synthase, which proceed with retention of configuration at the carbon center undergoing functionalization. (Chemical Equation Presented).