Biochemical and structural characterization of bisubstrate inhibitors of BasE, the self-standing nonribosomal peptide synthetase adenylate-forming enzyme of acinetobactin synthesis.
The human pathogen Acinetobacter baumannii produces a siderophore called acinetobactin that is derived from one molecule each of threonine, histidine, and 2,3-dihydroxybenzoic acid (DHB). The activity of several nonribosomal peptide synthetase (NRPS) enzymes is used to combine the building blocks into the final molecule. The acinetobactin synthesis pathway initiates with a self-standing adenylation enzyme, BasE, that activates the DHB molecule and covalently transfers it to the pantetheine cofactor of an aryl-carrier protein of BasF, a strategy that is shared with many siderophore-producing NRPS clusters. In this reaction, DHB reacts with ATP to form the aryl adenylate and pyrophosphate. In a second partial reaction, the DHB is transferred to the carrier protein. Inhibitors of BasE and related enzymes have been identified that prevent growth of bacteria on iron-limiting media. Recently, a new inhibitor of BasE has been identified via high-throughput screening using a fluorescence polarization displacement assay. We present here biochemical and structural studies to examine the binding mode of this inhibitor. The kinetics of the wild-type BasE enzyme is shown, and inhibition studies demonstrate that the new compound exhibits competitive inhibition against both ATP and 2,3-dihydroxybenzoate. Structural examination of BasE bound to this inhibitor illustrates a novel binding mode in which the phenyl moiety partially fills the enzyme pantetheine binding tunnel. Structures of rationally designed bisubstrate inhibitors are also presented.
Drake
Eric J
EJ
Hauptman-Woodward Institute and Department of Structural Biology, University at Buffalo, Buffalo, NY 14203-1102, USA.
Duckworth
Benjamin P
BP
Neres
João
J
Aldrich
Courtney C
CC
Gulick
Andrew M
AM
D040981:Acinetobacter baumannii / Q000201:enzymology*; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism*; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D010080:Oxazoles / Q000378:metabolism*; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D011485:Protein Binding
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D010080:Oxazoles; C091186:acinetobactin; D010453:Peptide Synthases