Uncoupled Epimerization and Desaturation by Carbapenem Synthase: Mechanistic Insights from QM/MM Studies

Abstract

Author: Carbapenem antibiotics possess a broad spectrum of antibacterial activity and high resistance to hydrolytic inactivation by β-lactamases. Carbapenem synthase (CarC), an iron(II) and 2-(oxo)glutarate-dependent oxygenase, catalyzes the epimerization and desaturation of (3S,5S)-carbapenam to produce (5R)-carbapenem in the last step of the simple carbapenem biosynthesis. Recently, the complete crystal structure of CarC was reported, allowing us to perform accurate quantum mechanics/molecular mechanics calculations to explore the detailed reaction mechanism. We first analyzed the dioxygen binding site on metal and identified that the FeIV–oxo species has two potential orientations with the oxo group trans to either His101 or His251. The former is energetically unstable, which can rapidly isomerize into the latter by rotation of the oxo group. Arg279 plays important roles in regulating the dioxygen binding and assisting the isomerization of FeIV–oxo species. The calculation results clearly support the stepwise C5-epimerization and C2/3-desaturation processes, involving two complete oxidative cycles. The epimerization process converts (3S,5S)-carbapenam to the initial product (3S,5R)-carbapenam, undergoing H5 atom abstraction by FeIV═O species, inversion of the C5-radical, and reconstitution of the inverted C5–H bond by Tyr165. In the desaturation process, (3S,5R)-carbapenam rebinds the CarC active site with a new orientation different from what (3S,5S)-carbapenam does in the epimerization. In addition, the desaturation across C2–C3 occurs without involving any active site residue other than the FeIV═O center. Whereas Tyr165 is not involved in the desaturation reaction, it plays a key role in binding (3S,5R)-carbapenam. (3S,5R)-Carbapenam is a substrate superior to its epimer (3S,5S)-carbapenam for CarC to produce (5R)-carbapenem by efficient desaturation. In addition, the substrate hydroxylations compete with the target epimerization and desaturation reaction.