A new robust kinetic assay for DAP epimerase activity

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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. Diaminopimelate decarboxylase (DAPDC)3 (E.C. 4.1.1.20) is a member of the pyridoxal 5Ј-phosphate (PLP)-dependent decarboxylases (1). It catalyzes the irreversible and stereospecific decarboxylation of meso-diaminopimelate (meso-DAP) in the final step of the diaminopimelate (DAP) biosynthesis pathway of bacteria and plants (2, 3). The product of the reaction, L-lysine, is an important building block for the biosynthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Consequently, DAPDC represents a promising target for the development of novel antibacterial agents (4).A DAPDC reaction mechanism has been previously proposed (5, 6). The reaction mechanism is thought to be initiated by the formation of a Schiff base between PLP and a conserved active site lysine from the (Y/F)AXKA motif (7). The substrate, meso-DAP, then binds and forms a subsequent Schiff base with PLP. This is suggested to be coordinated by the highly conserved CE(S/T)XD motif provided by an adjacent subunit (7). Decarboxylation of meso-DAP is then mediated by the sulfhydryl nucleophile provided by the conserved cysteine of the CE(S/T)XD motif thereby leaving the substrate cradled between two monomers (8). This proposed mechanism suggests DAPDC functions as a dimer.To support this assertion, previous structural studies of DAPDC from Methanocaldococcus jannaschii (PDB codes 1TWI and 1TUF) (9), Aquifex aeolicus (PDB code 2P3E), and Brucella melitensis (PDB code 3VAB) show that the enzyme crystallizes as a homodimer, with each monomer comprised of two domains (9). Domain I forms an ␣/␤ barrel, whereas domain II is comprised of a mixed ␤-sheet flanked by ␣-helices (9). The overall fold is similar to the functionally related enzyme ornithine decarboxylase, which also uses PLP as a cofactor in a decarboxylatio...

Section: Methodsmentioning

confidence: 99%

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Peverelli

Costa

Kirby

et al. 2016

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“…Protein samples were then concentrated to ϳ15-20 mg ml Ϫ1 and flash frozen in liquid nitrogen. Recombinant DAPDH protein was expressed and purified from E. coli BL21(DE3) (vector was a generous gift from Lilian Hor and Matthew Perugini, La Trobe University, Australia) as previously described (18).…”

Section: Ac-l-ala-iso-d-glu(oh)-meso-dap-oh (Tripeptide Substrate)-mentioning

confidence: 99%

Helical Shape of Helicobacter pylori Requires an Atypical Glutamine as a Zinc Ligand in the Carboxypeptidase Csd4

Chan

Blair

Liu

et al. 2015

“…Sedimentation velocity experiments were conducted in a Beckman model XL-A analytical ultracentrifuge at a temperature of 20°C using a similar method reported previously (37). Briefly, 380 l of sample (0.15 mg/ml) and 400 l of reference solution (10 mM phosphate, 137 mM NaCl, pH 7.4) were loaded into a conventional double sector quartz cell and mounted in a Beckman 4-hole An-60 Ti rotor.…”

Section: Analytical Ultracentrifugationmentioning

confidence: 99%

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4

Griffiths¹,

Dolezal

Cao

et al. 2016

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