FIG. 4.

SDS-PAGE and HPLC chromatograms of IptA assays. (A) SDS-12.5% PAGE analysis of purified His-tag-free IptA. Lane 1, molecular mass markers; lane 2, purified enzyme (arrow). The enzyme purification was described in Materials and Methods. Protein was stained with Coomassie brilliant blue R-250. (B) IptA (60 pmol enzyme) was incubated with l-Trp (0.5 mM) and DMAPP (0.2 mM). The enzyme reaction product was analyzed by LC-MS under the conditions described in Materials and Methods. Dotted and solid lines indicate product profiles after 1- and 10-min reactions, respectively. (C) IptA (100 pmol enzyme) was incubated with l-Trp (0.5 mM) and GPP (0.1 mM) for 120 min.