Investigations of the MceIJ-Catalyzed Posttranslational Modification of the Microcin E492 C-Terminus: Linkage of Ribosomal and Nonribosomal Peptides To Form “Trojan Horse” Antibiotics

Abstract

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.