Extended Data Figure 6. HPLC comparative analysis of the reactions catalysed by holo-module 5 and holo-M5*H790A with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP₄ (2).

a. Schematic of incubation of the tetrapeptidyl substrate L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP₄ (2) with holo-M5*H790A supplemented with ATP and L-pHPG. Pro-nocardicin G was not produced. b. HPLC comparison of unbound products from reaction mixtures containing L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP₄ (2) and either holo-module 5 or M5*H790A variant supplemented with ATP, L-pHPG, indicating the formation of β-lactam product only from the wild-type reaction. (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-PCP₄ (2), L-pHPG and ATP. (ii) HPLC trace of the unbound products resulting from incubation of holo-M5*H790A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP₄ (2), L-pHPG and ATP. Pro-nocardicin G was not observed. (iii) HPLC trace of the unbound products resulting from incubation of holo-M5*H792A reaction with L-pHPG-L-Arg-D-pHPG-L-Ser-S-PCP₄ (2), L-pHPG and ATP. (iv) HPLC trace of authentic standard of pro-nocardicin G.