Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Abstract

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...

Structure and Function of Cyanobacterial DHDPS and DHDPR

“…Sedimentation velocity experiments were performed at 20 °C in a Beckman Coulter Model XL-A analytical ultracentrifuge using double sector quartz cells and An50-Ti rotor4950. 400 μl of buffer and 380 μl of sample at an initial concentration ranging from 0.1 μM to 7.0 μM were loaded into the reference and sample sectors of the cells, respectively.…”

Crystal structure of a SFPQ/PSPC1 heterodimer provides insights into preferential heterodimerization of human DBHS family proteins

“…Sedimentation velocity experiments were conducted at 20°C using a Beckman Coulter XL-A or XL-I analytical ultracentrifuge with an An-50 Ti 8-hole rotor using similar methods reported previously (21,22). Briefly, double-sector quartz cells were loaded with 380 l of protein samples at an initial concentration of 5 M and 400 l of reference buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl).…”

“…Sedimentation equilibrium experiments were performed in a Beckman Coulter XL-I analytical ultracentrifuge in an An-50 Ti 8-hole rotor employing similar methods reported previously (21,22). Briefly, 120 l of protein samples (0.5-15 M) and 140 l of reference buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl) were centrifuged at rotor speeds of 12,000 and 18,000 rpm until sedimentation equilibrium was attained (t ϭ 36 and 48 h for each speed).…”

“…Experiments were carried out using a step size of 0.001 cm with 10 averages at a wavelength optimized at 234 nm. Data were fitted to a monomer-dimer equilibrium model using SEDPHAT (21,22,28).…”

Identification of two dihydrodipicolinate synthase isoforms from Pseudomonas aeruginosa that differ in allosteric regulation

“…1) [12,13,15,16]. 1) [12,13,19]. Gram-negative bacteria, including P. aeruginosa, are proposed to utilize the succinyl pathway to produce LL-DAP ( Fig.…”