Mechanism of the C5 Stereoinversion Reaction in the Biosynthesis of Carbapenem Antibiotics

Abstract

The bicyclic β-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of L-proline followed by β-lactam ring closure. Carbapenem synthase (CarC), an Fe(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.

Divergent non-heme iron enzymes in the nogalamycin biosynthetic pathway

“…3F) and suggests that amino acid radicals may not be involved in the catalytic cycle. In addition to critical structural differences, the two enzymes seem to use α-KG differently; although native CarC has been reported to display "one-third-of-sites reactivity" and alteration of α-KG utilization in CarC Y165F (13), the use of the cosubstrate remains comparable in SnoN and SnoN Y74F (Fig. S2J).…”

“…4). In CarC (Dali score Z = 8.2, sequence identity to SnoN 14%), the stereoinversion is completed by donation of a hydrogen atom from Tyr165 from the other side of the substrate (13). However, the active site cavity of SnoN contains only three amino acids that are likely to form and donate radicals (Trp64, Tyr74, and Trp180) (Fig.…”

“…Only one enzyme homologous to SnoN has been characterized to catalyze similar chemistry; the bifunctional CarC is responsible for stereoinversion and subsequent desaturation reactions in carbapenem biosynthesis (13). The mechanism of epimerization by CarC has been noted to involve ferryl abstraction of substrate hydrogen, followed by donation of a hydrogen atom from Tyr165, which effectually limits the enzyme activity to single turnover in vitro without a supply of external electrons (10).…”

“…We show that SnoK is responsible for an enzymatic carbocyclization whereas SnoN is an epimerase. The bifunctional α-KG-dependent CarC catalyzes an epimerization step in the biosynthesis of the antibiotic carbapenem (13), but SnoN displays striking structural and mechanistic differences. The findings thus expand the repertoire of reactions reported for this enzyme family and provide an illuminating example of divergent enzyme evolution where structurally similar proteins catalyze completely different chemistry.…”

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

“…The epimerization mechanism of CarC was subsequently modified and elaborated by Chang et al (77) (Fig. 3D), who overcame major obstacles including protein and substrate instability.…”