Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis.

Diaminopimelate decarboxylase (DAPDC) catalyzes the final step in the diaminopimelate biosynthesis pathway of bacteria. The product of the reaction is the essential amino acid l-lysine, which is an important precursor for the synthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Accordingly, the enzyme is a promising antibacterial target. Previous structural studies demonstrate that DAPDC exists as monomers, dimers, and tetramers in the crystal state. However, the active oligomeric form has not yet been determined. We show using analytical ultracentrifugation, small angle x-ray scattering, and enzyme kinetic analyses in solution that the active form of DAPDC from Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, and Vibrio cholerae is a dimer. The importance of dimerization was probed further by generating dimerization interface mutants (N381A and R385A) of V. cholerae DAPDC. Our studies indicate that N381A and R385A are significantly attenuated in catalytic activity, thus confirming that dimerization of DAPDC is essential for function. These findings provide scope for the development of new antibacterial agents that prevent DAPDC dimerization.

An optimized coupled assay for quantifying diaminopimelate decarboxylase activity.

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.

A new robust kinetic assay for DAP epimerase activity.

DAP epimerase is the penultimate enzyme in the lysine biosynthesis pathway. The most versatile assay for DAP epimerase catalytic activity employs a coupled DAP epimerase-DAP dehydrogenase enzyme system with a commercial mixture of DAP isomers as substrate. DAP dehydrogenase converts meso-DAP to THDP with concomitant reduction of NADP(+) to NADPH. We show that at high concentrations, accumulation of NADPH results in inhibition of DAPDH, resulting in spurious kinetic data. A new assay has been developed employing DAP decarboxylase that allows the reliable characterisation of DAP epimerase enzyme kinetics.