Figure 11.

Catalytic activity of mutated reductases.A and B, CE profiles at 30 min and kinetics for reductase activity on heptose whereby the substrates P4α, P4β, and P4γ were generated by DdahA and MlghB. A master reaction containing 0.37 mM of heptose, 0.5 mM of NADPH/⁺, and 0.4 μM MlghB was incubated for 30 min. For panel A, 0.1 μM (final concentration) of reductase was added to 9 μl of master mix and the volume brought to 10 μl before further incubation for 30 min. For panel B, the master mix was ultrafiltered to remove MlghB and used to perform the reductase kinetics on fixed amounts of P4α, P4β, and P4γ substrates incubated for up to 60 min with 0.5 μM (final concentration) of reductase. Reductase activity is denoted by formation of peaks P5α for DdahC or P5γ for MlghC from epimers P4α and P4γ, respectively. The data shown are from one experiment and are representative of independent repeats performed with different enzyme batches and showing similar trends. C and D, activity on GDP-mannose whereby the substrate P4’ was generated by HP0044 and MlghB. For panel C, reactions contained 0.3 mM of mannose, 0.3 mM of NADPH/⁺, 0.2 μM of HP0044, 0.4 μM of MlghB, and 0.5 μM of reductase in 10 μl and were incubated for 5 h. The data shown are representative of 2 independent repeats. For panel D, reactions contained 0.1 mM of mannose, 0.27 μM of HP0044, 0.34 mM of NADPH/⁺, 0.65 μM of epimerase MlghB, and 1 μM of reductase in 10 μl and were incubated for 1 h 45 mins. The different stoichiometry in panel D aimed at slowing down the formation of the epimer substrate to maximize its reduction by more abundant DdahC. The reactions were set in duplicates. and one representative trace is shown for each in panel D. For both panels C and D, complete usage of mannose substrate in the control reaction (MlghB trace, no reductase) ensures that the peak in the overlapping mannose/PII area of reductase-containing reactions is the reduction product PII.