No abstract

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1 H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K m for chorismate of 4.2 M and a k cat of 8 min ؊1 . The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1 H NMR spectroscopy.

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The reactive beta-ketoacid pyridoxal-5'-phosphate aldimine formed in the condensation step of the 8-amino-7-oxononanoate synthase reaction was 'trapped' in the enzyme-bound form by carrying out the reaction with l-alanine methyl ester and pimeloyl-CoA affording the more stable methyl ester of the putative intermediate, the characterisation of which provides the first definitive evidence for a beta-ketoacid intermediate in an alpha-oxamine synthase mechanism.

The in silico design, synthesis, and biological evaluation of ten potent type II dehydroquinase inhibitors are described. These compounds contain an anhydroquinate core, incorporated as a mimic of the enolate reaction intermediate. This substructure is attached by a variety of linking units to a terminal phenyl group that binds in an adjacent pocket. Inhibitors were synthesised from (-)-quinic acid using palladium-catalysed Stille and carboamidation chemistry. Several inhibitors exhibited nanomolar inhibition constants against type II dehydroquinases from Streptomyces coelicolor and Mycobacterium tuberculosis. These are among the most potent inhibitors of these enzymes reported to date.