Fig. 7.

Structure-guided mutagenesis. (A) Aliquots (8 μg) of the Ni-agarose preparations of the indicated His₁₀CthPnkp-(462–870) proteins (purified as described in Supplemental Information) were analyzed by SDS-PAGE. The polypeptides were visualized by staining with Coomassie blue dye. The positions and sizes (kDa) of marker polypeptides are indicated. (B) Adenylyltransferase activity. Reaction mixtures (20 μL) containing 50 mM Tris-acetate (pH 7.0), 5 mM DTT, 5 mM MgCl₂, 20 μM [α³²P]ATP, and 2 μg of wild-type or mutated His₁₀CthPnkp were incubated at 37 °C for 15 min. The reactions were quenched with SDS and the products were analyzed by SDS-PAGE. The enzyme-[³²P]AMP adduct was quantified by scanning the gel with a Fujix imager. The extents of label transfer from [α³²P]ATP to the respective CthPnkp-(462–870) polypeptides are shown. Each datum in the bar graphs is the average of three separate experiments ± SEM.