Extended Data Figure 3. Negative experiments resulting from incubation of holo-module 5 with alternate substrates.

a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) with holo-module 5 supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC traces of products obtained after incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. Pro-nocardicin G was not observed as verified by comparison to authentic standard. Additionally, it was noted that substrate 3 was not consumed over the duration of the experiment. (i) HPLC trace of the unbound products resulting from incubation of wild-type holo-module 5 reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP. (iii) HPLC trace of incubation of L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3), L-pHPG and ATP without enzyme. The peak corresponding to L-pHPG-L-Arg-D-pHPG-L-Ser-S-pantetheine (3) substrate is indicated. (iv) HPLC trace of authentic standard of pro-nocardicin G. c. Schematic of incubation of the dipeptidyl substrate D-pHPG-L-Ser-S-PCP₄ (5) with holo-module 5, L-pHPG and ATP. Nocardicin G, the corresponding expected product, was not observed. d. LC-MS traces of products obtained after incubation of D-pHPG-L-Ser-S-PCP₄ (5) and holo-module 5 [+M5(wt)] supplemented with ATP and L-pHPG. The corresponding β-lactam product was not observed as verified by comparison to authentic standard. (i) Total ion chromatogram of the unbound products resulting from wild-type holo-module 5 reaction with D-pHPG-L-Ser-S-PCP₄ (5), L-pHPG and ATP (ii) Extracted ion chromatogram of the wild-type reaction in trace i, the 386.1 m/z ion, corresponding to [M+H] of nocardicin G was not observed. (iii) Total ion chromatogram of unbound products from holo-M5*H792A control reaction with D-pHPG-L-Ser-S-PCP₄ (5), L-pHPG and ATP. (iv) Extracted ion chromatogram of the wild-type reaction in trace iii. (v) Extracted ion chromatogram of authentic standard of nocardicin G.