FIGURE 4.
CE electrophoregrams highlighting the differences between the reductases WcaGNCTC and WcaG81176. The identity of all peaks is as described in Fig. 3. In addition, P3 is reduced GDP-6-deoxy-manno-heptose. Panel A, ∼0.10 mm preformed P1, 7 μm MlghB, 0.10 mm NADPH, and 200 mm Tris-HCl, pH 8.0 were incubated at 37 °C for 15 min, and the reaction was filtered through a 10-kDa membrane to remove MlghB. The filtrate obtained comprised products P1, P4α, P4β, and P4γ in fixed proportions. It was incubated for 30 min at 37 °C as such (trace a) or supplemented with 3 μm concentrations each of the indicated enzymes. The data show that WcaGNCTC and WcaG81176 have the same activity and cannot replace the reductase of their cognate pathway, MlghC or DdahC. Panel B, 6 μl of ∼0.07 mm heptose and 3 μm DdahA (trace a, base line) were supplemented with 3 μm concentrations of each of the indicated enzymes (without elimination of DdahA). The activity was recorded after 2.5 h incubation at 37 °C. The data show that the activity of MlghC is not affected by WcaGNCTC and vice versa. Integration data are provided in supplemental Table S2.