Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution - PubMed (original) (raw)

Plate-based enzyme assay. A, endogenous phosphatase activity in crude lysate. Sugar 1-phosphate (2 m

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) (filled circles, Glc 1-phosphate; open circles, Gal-1-phosphate; filled triangles, Man 1-phosphate; open triangles, GlcNAc 1-phosphate) was reacted with BL21(DE3) crude lysate (at 2.5× concentration with respect to culture volume) in the absence of nucleotide. Without the addition of alkaline phosphatase, the reacted samples were tested directly for the presence of reducing sugar with the pHBH reagent in the plate form assay. B, comparison of solution phase and solid phase activities of WT RmlA (filled circles, 5 m

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nominal RmlA on resin; open circles, 5 m

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RmlA in solution; filled triangles, 1.7 m

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RmlA in solution). C, the influence of Ni2+-NTA-agarose (filled circles, titration without resin; open circles, titration with resin) and EDTA (filled triangles, with resin in the presence of 25 m

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EDTA) on pHBH reaction sensitivity. D, representative on-resin plate-based assay for RmlA variants screened against galactose 1-phosphate and UTP. The average activities of parent enzyme (WT RmlA) replicates (dotted line) are well separated from empty vector controls (dashed line). Wells with activity slightly greater than wild type are readily identifiable.