title: Carbapenem biosynthesis: confirmation of stereochemical assignments and the role of CarC in the ring stereoinversion process from L-proline.
abstract: (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.
authors.affiliation: Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Stapon
authors.forename: Rongfeng
authors.initials: R
authors.lastname: Li
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: 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
chemical_list: D000900:Anti-Bacterial Agents; D015780:Carbapenems; D007769:Lactams; C057567:carbapenam-3-carboxylic acid; D011392:Proline