FIGURE 2.

In vitro reconstitution of petrobactin biosynthesis. Heterologously expressed asb products were combined with metabolic precursors spermidine, citric acid, and 3,4-DHBA and the necessary substrate ATP and cofactor Mg²⁺. A, LC-MS data demonstrates accumulation of m/z = 360.3 over time, corresponding to the [M+2H]²⁺ of petrobactin. The compound associated with this peak was analyzed by MS/MS and shown to have a fragmentation pattern identical to that of authentic petrobactin. B, products from various combinations of asb enzymes were investigated by LC-MS. To detect zwitterionic intermediates generated by the NIS synthetases AsbA and AsbB, reactions were acid quenched, and products were derivatized with fluorescamine (top two traces). Other organic products were extracted with methanol for analysis (lower traces). Omission of AsbA still results in a modest accumulation of petrobactin, suggesting a compensatory role is filled by the type C NIS synthetase AsbB. Traces show relative intensity of selected m/z for each compound.