The carbapenem class of beta-lactam antibiotics is known for its remarkable potency, antibacterial spectrum, and resistance to beta-lactamase-mediated inactivation. While the biosynthesis of structurally "complex" carbapenems, such as thienamycin, share initial biochemical steps with carbapenem-3-carboxylate ("simple" carbapenem), the requisite inversion at CS and formation of the characteristic alpha,beta-unsaturated carboxylate are different in origin between the two groups. Here, we consider carbapenem synthase, a mechanistically distinct bifunctional non-heme iron alpha-ketoglutarate-dependent enzyme responsible for the terminal reactions, CS epimerization and desaturation, in simple carbapenem production. Interestingly, this enzyme accepts two stereoisomeric substrates and transforms each to a common active antibiotic. Owing both to enzyme and product instability, resorting to saturation mutagenesis of active site and selected second-sphere residues gave clearly differing profiles of CarC tolerance to structural modification. Guided by a crystal structure and the mutational data, in silico docking was used to suggest the positioning of each disastereomeric substrate in the active site. The two orientations relative to the reactive iron-oxo center are manifest in the two distinct reactions, C5-epimerization and C2/3-desaturation. These observations favor a two-step reaction scheme involving two complete oxidative cycles as opposed to a single catalytic cyde in which an active site tyrosine, Tyr67, after hydrogen donation to achieve bicyclic ring inversion, is further hypothesized to serve as a radical carrier.