Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

Abstract

Andrimid is a hybrid nonribosomal peptide-polyketide antibiotic that blocks the carboxyl-transfer reaction of bacterial acetyl-CoA carboxylase (ACC) and thereby inhibits fatty acid biosynthesis with submicromolar potency. The andrimid biosynthetic gene cluster from Pantoea agglomerans encodes an admT gene with homology to the acetyl-CoA carboxyltransferase (CT) ␤-subunit gene accD. Escherichia coli cells overexpressing admT showed resistance to andrimid. Co-overproduction of AdmT with E. coli CT ␣-subunit AccA allowed for the in vitro reconstitution of an active heterologous tetrameric CT A 2T2 complex. A subsequent andrimidinhibition assay revealed an IC 50 of 500 nM for this hybrid A2T2 in contrast to that of 12 nM for E. coli CT A 2D2. These results validated that AdmT is an AccD homolog that confers resistance in the andrimid producer. Mutagenesis studies guided by the x-ray crystal structure of the E. coli A 2D2 complex disclosed a single amino acid mutation of AdmT (L203M) responsible for 5-fold andrimid sensitivity (IC 50 ‫؍‬ 100 nM). Complementarily, the E. coli AccD mutant M203L became 5-fold more resistant in the CT assays. This observation allowed for bioinformatic identification of several Vibrio cholerae strains in which accD genes encode the Met7Leu switches, and their occurrences correlate predictively with sensitivities to andrimid in vivo.antibiotic resistance ͉ fatty acid biosynthesis inhibitor

Slow Onset Inhibition of Bacterial β-Ketoacyl-acyl Carrier Protein Synthases by Thiolactomycin

“…This assay system was used to measure the IC 50 value of TLM with ecFabF. Unlike KasA, ecFabF showed no change in IC 50 upon preincubation, and our value of 5.4 Ϯ 0.5 M (Table 3) is consistent with the previously published value of 6 M (16). These data suggest that TLM is not a slow binding inhibitor of ecFabF.…”

“…Finally, although the slow onset inhibition of smFabB and kpFabB presents a plausible explanation for the in vivo antibacterial activity of TLM against S. marcescens and K. pneumonia, it may also be that the inherent resistance of the TLM producer species Nocardia and Streptomyces to TLM results from the reduced affinity and/or absence of slow onset inhibition of the antibiotic for the enzyme target(s) in these organisms, as recently shown in an analogous fashion for resistance of Pantoea agglomerans to the antibiotic andrimid (50).…”

“…Subsequently IC 50 values were determined by first preincubating TLM and KasA followed by initiation of the reaction with one or other of the two substrates ( Table 2). The IC 50 value determined when palmitoyl-AcpM was used to initiate the preincubated reaction mixture containing KasA, TLM, and SCHEME 1. General scheme for the two-step formation of an enzyme-inhibitor complex.…”

“…The kinetic parameters for ecFabF (500 nM) were determined in 50 mM potassium phosphate buffer, pH 6.5, containing 50 mM NaCl, 1 mM dithiothreitol by varying the concentration of lauroyl-CoA at a fixed concentration of malonyl-CoA (2 mM) or by varying the concentration of malonyl-CoA at a fixed concentration of lauroyl-CoA (100 M). The IC 50 for the inhibition of ecFabF with TLM was determined using this assay system by varying the concentration of TLM at fixed concentrations of lauroyl-CoA (100 M) and malonyl-CoA (550 M). All kinetic data were analyzed using Grafit 4.0.…”

Dimerization of the Bacterial Biotin Carboxylase Subunit Is Required for Acetyl Coenzyme A Carboxylase Activity In Vivo

“…The subunits can readily be purified to study the partial reactions. Each of the acc genes is essential for growth of E. coli (2), and ACC is a validated antibiotic target (9,21).…”