ABSTRACT
IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7â Å resolution, respectively. The structure of the E.â coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Design , Enzyme Inhibitors/pharmacology , Erythritol/analogs & derivatives , Escherichia coli Proteins/chemistry , Escherichia coli/drug effects , Escherichia coli/enzymology , Oxidoreductases/chemistry , Sugar Phosphates/metabolism , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Erythritol/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/metabolism , Molecular Docking Simulation , Molecular Structure , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/metabolismABSTRACT
The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.
Subject(s)
Enzyme Inhibitors/pharmacology , Erythritol/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/metabolism , Oxidoreductases/antagonists & inhibitors , Oxidoreductases/metabolism , Sugar Phosphates/pharmacology , Terpenes/metabolism , Biocatalysis , Biosynthetic Pathways/drug effects , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Erythritol/analogs & derivatives , Erythritol/chemistry , Escherichia coli Proteins/isolation & purification , Kinetics , Models, Molecular , Molecular Structure , Oxidoreductases/isolation & purification , Structure-Activity Relationship , Sugar Phosphates/chemical synthesis , Sugar Phosphates/chemistry , Terpenes/chemistryABSTRACT
The MEP pathway for the biosynthesis of isoprene units is present in most pathogenic bacteria, in the parasite responsible for malaria, and in plant plastids. This pathway is absent in animals and is accordingly a target for the development of antimicrobial drugs. LytB, also called IspH, the last enzyme of this pathway catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into a mixture of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) using an oxygen sensitive iron sulfur cluster. The exact nature of this iron sulfur cluster is still a matter of debate. We have used (57)Fe Mössbauer spectroscopy to investigate the LytB cluster in whole E. coli cells and in the anaerobically purified enzyme: In LytB an unusual [4Fe-4S](2+) cluster is attached to the protein by three conserved cysteines and contains a hexacoordinated iron linked to three sulfurs of the cluster and three additional oxygen or nitrogen ligands.