Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.

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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...

Fluoride is everywhere in the environment, yet it is toxic to living things. How biological organisms detoxify fluoride has been unknown until recently. Fluoride-specific ion transporters in both prokaryotes (Fluoride channel; Fluc) and fungi (Fluoride Exporter; FEX) efficiently export fluoride to the extracellular environment. FEX homologues have been identified throughout the plant kingdom. Understanding the function of FEX in a multicellular organism will reveal valuable knowledge about reducing toxic effects caused by fluoride. Here we demonstrate the conserved role of plant FEX (FLUORIDE EXPORTER) in conferring fluoride tolerance. Plant FEX facilitates the efflux of toxic fluoride ions from yeast cells and is required for fluoride tolerance in plants. A CRISPR/Cas9-generated mutation in Arabidopsis thaliana FEX renders the plant vulnerable to low concentrations (100 µM) of fluoride at every stage of development. Pollen is particularly affected, failing to develop even at extremely low levels of fluoride in the growth medium. The action of the FEX membrane transport protein is the major fluoride defense mechanism in plants.

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