Diaminopimelate decarboxylase catalyzes the final step in lysine biosynthesis in bacteria. The epimerase catalyzes the interconversion of the ll- and meso isomers of diaminopimelate. Because these enzymes are absent in mammals, they are considered to be potential targets for antimicrobial agents.
The o-phthaldialdehyde derivatives of ll-diaminopimelate, meso-diaminopimelate, norvaline (internal standard), and lysine were separated on a Spherisorb Ci8 column (4.5 mm x 250 mm). A linear gradient from 100% solvent A (30 methanol, 70% 50 mAi sodium acetate buffer (pH 5.9) to 30% solvent A and 70% methanol was imposed over 35 minutes. Detection was by fluorescence, with excitation and emission wavelength of 340 and 455 nm, respectively.
The normal incubation mixture contained in 0.1 M potassium phosphate (pH 7.0) in a final volume of 1.5 mL: 15 /¿mol recrystallized meso-diaminopimelate, 0.1 /xmol pyridoxal 5-phosphate, 3.75 /xmol norvaline, and desalted cell-free extract (about 0.5 mg protein). The reaction was carried out at 37°C and was started by adding the extract. Aliquots of 500 /xL were taken immediately before and after the 15-minute incubation period and transferred to 10 mL Pyrex tubes held in a boiling water bath, each containing 1.0 mL sodium acetate buffer (1.2 M, pH 5.2). After heating for 5 minutes, the tubes were centrifuged and an aliquot of the supernate was diluted 20-fold with water. The derivatization reaction was carried out by mixing 200 fiL of the diluted supernate with 200 fiL of a sodium dodecyl sulfate solution (2%, w/ v, in 400 mM sodium borate buffer, pH 9.5), and adding 400 fiL of the o-phthaldialdehyde/2-mercaptoethanol derivatizing solution. After exactly 1 minute of derivatization, an aliquot was injected onto the HPLC column.
The source of enzyme was cell-free extracts prepared from a lysine-overproducing strain of Bacillus subtilis NCIB 3610.
Figure 9.51 shows representative chromatograms.
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