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An optimized coupled assay for quantifying diaminopimelate decarboxylase activity.

Biochimie (2015-05-20)
Martin G Peverelli, Matthew A Perugini
ABSTRACT

Diaminopimelate decarboxylase (DAPDC) catalyzes the conversion of meso-DAP to lysine and carbon dioxide in the final step of the diaminopimelate (DAP) pathway in plants and bacteria. Given its absence in humans, DAPDC is a promising antibacterial target, particularly considering the rise in drug-resistant strains from pathogens such as Escherichia coli and Mycobacterium tuberculosis. Here, we report the optimization of a simple quantitative assay for measuring DAPDC catalytic activity using saccharopine dehydrogenase (SDH) as the coupling enzyme. Our results show that SDH has optimal activity at 37 °C, pH 8.0, and in Tris buffer. These conditions were subsequently employed to quantitate the enzyme kinetic properties of DAPDC from three bacterial species. We show that DAPDC from E. coli and M. tuberculosis have [Formula: see text] of 0.97 mM and 1.62 mM and a kcat of 55 s(-1) and 28 s(-1), respectively, which agree well with previous studies using more labor-intensive assays. We subsequently employed the optimized coupled assay to show for the first time that DAPDC from Bacillus anthracis possesses a [Formula: see text] of 0.68 mM and a kcat of 58 s(-1). This optimized coupled assay offers excellent scope to be employed in high throughput drug discovery screens targeting DAPDC from bacterial pathogens.

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