*4.5. Preparation of KDPG*

KDPG was prepared from the KDG by using recFlKin. Namely, recFlKin was (final concentration 10 μg/mL) was added to the reaction mixture (10 mL) containing 2.5 mM KDG, 2.5 mM ATP, 5 mM MgCl2, 20 mM Tris-HCl (pH 7.4), 100 mM KCl, and 1 mM dithiothreitol, and incubated at 40 ◦C for 3 h. The mixture was lyophilized, dissolved in 500 μL of distilled water and the supernatant was subjected to a Superdex peptide 10/300 GL column equilibrated with 0.1 M CH3COONH4. KDPG and KDG, which eluted together in this chromatography, were lyophilized, dissolved in 1 mL of distilled water, and subjected to HPLC (Shimadzu Prominence LC-6AD, Tokyo, Japan) equipped by TSKgel DEAE-2SW (Tosoh). KDG and KDPG were separately eluted at around 150 mM and 320 mM CH3COONH4 by the linear gradient of 0–0.4 M CH3COONH4. The amount of KDPG was quantified by the system comprising recFlAld and LDH–NADH using authentic KDPG as a standard. By the above procedure, 1.2 mg of KDPG was obtained from 4.5 mg of KDG.

#### *4.6. Assay for KDPG Aldolase Activity*

KDPG aldolase activity of recFlAld was assayed by the determination of pyruvate using a lactate dehydrogenase (LDH)–NADH coupling system [50]. Namely, the aldolase reaction was conducted at 30 ◦C in a reaction mixture containing 5 mM KDPG, 20 mM Tris-HCl (pH 7.4), 100 mM KCl, 1 mM DTT, and 1 μg/mL recFlAld in the presence of 0.2 mM NADH and 1 unit/mL LDH. The reaction rate was estimated from the decrease in the Abs 340 nm due to the oxidation of NADH accompanied by the reduction of pyruvate. One unit (U) of KDPG aldolase activity was defined as the amount of enzyme that produced 1 μmol of pyruvate per min.
