Figure 6.

Mg²⁺ concentration affects the activity of E. coli K-12 WaaO. Hep₂-1-deP-KLA (labeled A) was reacted with WaaG (0.01 mg/mL) for 15 min to first produce Glc-Hep₂-1-deP-KLA (labeled B). WaaB (0.1 mg/mL) was then added and incubated for an additional 135 min to produce Gal-Glc-Hep₂-1-deP-KLA (labeled C). WaaO was added (0.1 mg/mL) in the presence of various concentrations of Mg²⁺ as indicated after WaaB reacted as indicated. When Mg²⁺ was excluded, no WaaO-dependent glycosylation was detected. However, when Mg²⁺ was included, a product with migration consistent with glucose addition was detected. At a low Mg²⁺ concentration (0.1 mM), one product is detected (labeled D), but as the Mg²⁺ concentration is increased, multiple products (labeled D*) are detected, consistent with the addition of multiple glucoses to Gal-Glc-Hep₂-1-deP-KLA.