(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.
(5R)-Carbapenem-3-carboxylic acid is the simplest structurally among the naturally occurring carbapenem beta-lactam antibiotics. It is the produced from (3S,5S)-carbapenam-3-carboxylic acid utilizing a remarkable stereoinversion/desaturation process by CarC (carbapenem synthase), an alpha-ketoglutarate dependent non-heme iron oxygenase. In this communication, we demonstrate for the first time that the epimeric (3S,5R)-carbapenam-3-carboxylic acid is an intermediate in the overall catalytic cycle to the carbapenem antibiotic. The role of alpha-ketoglutarate in the stereoinversion and desaturation processes is also examined.
The 5-methyl-2-pyrrolylcarbonyl moiety of the aminocoumarin antibiotics clorobiocin and coumermycin A1 is the key pharmacophore for targeting the ATP-binding site of GyrB for inhibition of the bacterial type-II topoisomerase DNA gyrase. During the late stage of clorobiocin and coumermycin A1 biosynthesis, the pyrrolyl-2-carboxyl group is transferred from the peptidyl carrier proteins Clo/CouN1 to the 3'-hydroxyl of the 4-methoxy-L-noviosyl scaffold by the action of the acyltransferases Clo/CouN7. CouN1 and CouN7 have now been heterologously expressed and purified from Escherichia coli. The apo form of CouN1 is converted to the acyl-holo form by loading with pyrrolyl-2-carboxyl-S-pantetheinyl moieties from synthetic pyrrolyl- and 5-methylpyrrolyl-CoAs by the action of the phosphopantetheinyl transferase Sfp. CouN7 acts as an acyltransferase, moving the pyrrole acyl moieties from CouN1 to the noviose sugar of descarbamoylnovobiocin. When the 5-methylpyrrolyl-2-carboxyl-thioester of CouN1 is the cosubstrate, the in vitro product differs from clorobiocin only in a CH3 for Cl group change on the coumarin ring. Double transfer of this acyl moiety by CouN7 to the penultimate intermediate in coumermycin A1 assembly completes that antibiotic biosynthetic pathway.
Long-chain lipid envelopes are characteristic of mycobacteria such as those that cause tuberculosis and leprosy. Inhibition of fatty acid synthesis or elongation is a strategy demonstrated to be clinically effective against M. tuberculosis. A new class of compounds designed to inhibit the beta-ketoacyl synthase reaction of fatty acid synthesis has been developed. Of >30 compounds described, the most active were acetamides containing alkylsulfonyl substituents. Inhibitory activities were acutely sensitive to net charge, chain length, and degree of unsaturation. The most active compound 5 (alkyl = C(10)) contained a single methylene spacer between the sulfone and carboxamide and exhibited an MIC of 0.75-1.5 microg/mL, comparable to first-line antituberculosis drugs. These compounds are species-specific, exhibiting no significant activity against bacterial species other than M. tuberculosis and closely related strains. The synthesis, biological activity, and specificity of these compounds are described.
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