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Figure 5
WcbI shows no detectable acetyltransferase activity. (a) Acetyltransferase activity was measured using the DTNB assay. Each column represents an average of three measurements. The concentrations of acetyl-CoA and GDP-mannose were held constant at 1 and 4 mM, respectively. The same assay performed with chloramphenicol acetyltransferase and 2 mM chloramphenicol served as a positive control. Reactions in which recombinant WcbI and CAT were omitted served as a negative control. (b) Acetyltransferase activity was measured using a fluorescence-based activity assay. Concentrations of GDP-mannose ranging from 0 to 3 mM were used, with the WcbI concentration held constant. The positive control supplied in the kit was used according to the manufacturer's instructions. (c) Acetyltransferase activity was measured using a coupled assay with pyruvate dehydrogenase. Substrate concentrations were 0.5 mM for acetyl-CoA and 1 mM for GDP-mannose. The same assay was performed using CAT and 1 mM chloramphenicol as a positive control. A negative control was performed without either enzyme. The rates were calculated using the extinction coefficient for NADH. All columns represent the average ± standard deviation of two measurements.

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