ABSTRACT
We developed a homogenous microtiter based assay using the cationic dye 3, 3'-Diethyloxacarbocyanine iodide, DiOC2(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteriological Techniques/methods , High-Throughput Screening Assays/methods , Membrane Potentials/drug effects , Staphylococcus aureus/drug effects , Carbocyanines/metabolism , Escherichia coli/drug effects , Inhibitory Concentration 50ABSTRACT
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a member of the family of acyl-condensing enzymes, catalyzes the first committed step in the mevalonate pathway and is a potential target for novel antibiotics and cholesterol-lowering agents. The Staphylococcus aureus mvaS gene product (43.2 kDa) was overexpressed in Escherichia coli, purified to homogeneity, and shown biochemically to be an HMG-CoA synthase. The crystal structure of the full-length enzyme was determined at 2.0-A resolution, representing the first structure of an HMG-CoA synthase from any organism. HMG-CoA synthase forms a homodimer. The monomer, however, contains an important core structure of two similar alpha/beta motifs, a fold that is completely conserved among acyl-condensing enzymes. This common fold provides a scaffold for a catalytic triad made up of Cys, His, and Asn required by these enzymes. In addition, a crystal structure of HMG-CoA synthase with acetoacetyl-CoA was determined at 2.5-A resolution. Together, these structures provide the structural basis for an understanding of the mechanism of HMG-CoA synthase.
