FIGURE 6.

S-DAPAT-catalyzed reaction. A, scheme of the reaction. B, HPLC analyses of the reaction. The t₀ reaction mix (100 μl) contained 100 μm S-DAP and 100 μm α-ketoglutarate in 50 mm potassium phosphate buffer (pH 6.9). The reaction was initiated with the recombinant S-DAPAT (Sav_Q82IK5, Mtu_O50434, or Neu_Q82S89). The reactions were allowed to proceed for 15 min at 30 °C and were stopped by centrifugation on Vivaspin 10K concentration units. A 20-μl aliquot of the flow-through was derivatized for 1 min with o-phtaldialdehyde prior to its injection on a Spherisorb ODS2 C18 column connected to an Agilent 1100 HPLC system. Elution was carried out at 1 ml/min with a 10–60% methanol gradient (15 min) in 50 mm potassium phosphate buffer (pH 6.9). The o-phtaldialdehyde derivatives of amino acids were detected by fluorescence (excitation, 360 nm; emission, 455 nm). Glutamate was identified by comparison with an authentic standard.