Carbapenem biosynthesis: confirmation of stereochemical assignments and the role of CarC in the ring stereoinversion process from L-proline.
(5R)-Carbapen-2-em-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem beta-lactam antibiotics. It co-occurs with two saturated (3S,5S)- and (3S,5R)-carbapenam carboxylic acids. Confusion persists in the literature about the signs of rotation and absolute configurations of these compounds that is resolved in this paper. (3S,5S)-Carbapenam carboxylic acid was prepared from L-pyroglutamic acid to unambiguously establish its absolute configuration as identical to the natural product isolated from Serratia marcescens and from overexpression of the biosynthetic genes carAB in Escherichia coli. L-Proline labeled with deuterium or tritium at the diastereotopic C-5 methylene loci was shown to incorporate one label at the bridgehead of (3S,5S)-carbapenam carboxylic acid, but not into the "inverted" (3S,5R)-carbapenam carboxylic acid or the final carbapenem product. CarC, the third enzyme of the biosynthetic pathway required to synthesize the carbapenem, was demonstrated in cell-free studies to be dependent on alpha-ketoglutarate and ascorbate in keeping with weak sequence identities with other non-heme iron, alpha-ketoglutarate-dependent oxygenases. CarC mediated the stereoinversion of synthetic (3S,5S)-carbapenam carboxylic acid to the (5R)-carbapenem as judged by bioassay. These findings suggest that L-proline is desaturated to pyrroline-5-carboxylic acid prior to uptake into the biosynthetic pathway. The loss of the bridgehead hydrogen from the (3S,5S)-carbapenam during the ring inversion process to form the epimeric (3S,5R)-carbapenam and desaturation to the (5R)-carbapenem are proposed to be coupled by CarC to the reduction of dioxygen to drive the formation of these higher energy products, an unprecedented reaction for this enzyme class.
Stapon
Anthony
A
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Li
Rongfeng
R
Townsend
Craig A
CA
D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000737:chemistry; D015780:Carbapenems / Q000138:chemical synthesis* / Q000737:chemistry; D007769:Lactams; D008968:Molecular Conformation; D011392:Proline / Q000737:chemistry*; D013237:Stereoisomerism
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D007769:Lactams; C057567:carbapenam-3-carboxylic acid; D011392:Proline