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 CC 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'-H-2](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.