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. Author manuscript; available in PMC: 2015 Oct 16.
Published in final edited form as: Nature. 2015 Jan 26;520(7547):383–387. doi: 10.1038/nature14100

Figure 3. Analysis of the reactions catalysed by module 5.

Figure 3

a, Substrates used in this study. b, Incubation of tetrapeptidyl-S-PCP4 2 with holo-module 5, ATP and L-pHPG produced β-lactam containing pro-nocardicin G. c, Left: HPLC traces of products obtained after incubation of tetrapeptidyl-S-PCP4 2 and indicated construct, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H792A), verified by comparison to synthetic standard (top trace). Right: LC-MS traces of products obtained after incubation of tetrapeptidyl-S-PCP4 2 and holo-module 5, ATP and L-pHPG. Pro-nocardicin G was observed in the wild-type reaction [+M5(wt)] but not in the mutant (+M5*H792A), verified by comparison to synthetic standard (top trace). BPI = base peak ion, TOF = time of flight, ES = electrospray ionization, EI = extracted ion. Calculated exact mass of pro-nocardicin G = 691.2835 [M+H]+. d, Left: Time-course study of tetrapeptidyl-S-PCP4 2 and holo-module 5 supplemented with L-pHPG and ATP. Appearance of pro-nocardicin G was analyzed by HPLC. Right: Plots of pro-nocardicin G production (HPLC) and the corresponding conversion of 2 to the unloaded holo-PCP4, (ESI-MS). Less than 10% hydrolysis of 2 was observed during the 10-h experiment.