FIG 3.

The alanine scanning mutagenesis of critical residues in the ChPPK. (A) The crystal structure of ChPPK in complex with polyP₅ and AMP. The residues selected for alanine scanning mutagenesis, polyP₅ and AMP, were highlighted in the close-up stereo view of the catalytic pocket. (B) The relative activities of purified wild-type ChPPK and its variants. Enzymatic activity assays were performed for the purified enzymes (5 µg/mL) of ChPPK or its variants in the presence of 1.6 g/L PPA and 2.25 mM AMP. The ATP concentration after incubation was measured by HPLC. One unit of enzyme activity was given as the amount of enzyme that synthesized 1 µM ATP from AMP per minute during the initial minute.