(A) The dapdiamide family of antibiotics. (B) The dapdiamide gene cluster. (C) Dapdiamide-related epoxide natural products.

Apo-DdaD is converted to holo-DdaD (HS-DdaD) in the presence of Sfp and CoA. In the presence of ATP and N_βFmmDAP, HS-DdaD is loaded with N_βFmmDAP to form N_βFmmDAP-S-DdaD. (A) In the presence of Sfp but the absence of CoA, only the active site peptide from apo-DdaD (green) is observed. (B) In the presence of Sfp and CoA, the active site peptide from HS-DdaD (red) is observed. This peptide shows a mass shift of +340.09 Da from the apo-DdaD active site peptide, the exact mass shift expected for PPTation. (C) The small molecule Ppant ejection ion (red) is observed from the HS-DdaD active site peptide when the peptide is subjected to MS/MS by NSD. (D) Both predicted Ppant ejection peptide marker ions are observed when the HS-DdaD active site peptide is subjected to MS/MS using CID. (E) In the presence of ATP but the absence of N_βFmmDAP, only the HS-DdaD active site peptide (red) is observed. (F) In the presence of both ATP and N_βFmmDAP, both the HS-DdaD (red) and N_βFmmDAP-S-DdaD (blue) active site peptides are observed. (G) When the HS-DdaD and N_βFmmDAP-S-DdaD active site peptides are subjected to MS/MS using NSD, both expected small molecule Ppant ejection ions are observed. The HS-DdaD Ppant ejection ion is shown in red and the N_βFmmDAP-S-DdaD Ppant ejection is shown in blue. For the predicted structures of the Ppant ejection ions, see Figure S6.

In an atmosphere of ¹⁸O₂, ¹⁸O is incorporated into the substrate loaded onto DdaD and N_βFmmDAP is converted to N_β-trans-EpSmDAP. (A) Under normal reaction conditions (an atmosphere of ¹⁶O₂), in the presence of α-KG and the absence of DdaC, the only Ppant ejection ion observed (blue) during Ppant ejection analysis (using NSD) of N_βFmmDAP-S-DdaD corresponds to the unoxidized starting substrate. (B) Under normal reaction conditions (an atmosphere of ¹⁶O₂), in the presence of both α-KG and DdaC, Ppant ejection ions corresponding to both N_βFmmDAP-S-DdaD and N_βEpSmDAP-S-DdaD (orange) are observed. (C) Under an atmosphere of ¹⁸O₂, in the presence of α-KG but the absence of DdaC, the only Ppant ejection ion observed corresponds to N_βFmmDAP-S-DdaD. (D) Under an atmosphere of ¹⁸O₂, in the presence of both α-KG and DdaC, Ppant ejection ions are observed for both N_βFmmDAP-S-DdaD and N_βEpSmDAP-S-DdaD. A +2 Da mass shift (from the N_βEpSmDAP-S-DdaD Ppant ejection ion in (B)) is observed in the Ppant ejection ion corresponding to N_βEpSmDAP-S-DdaD (purple), indicating that the oxygen in the epoxide originates from molecular oxygen. For the predicted structures of the Ppant ejection ions see Figure S6.

DdaD ATP-[³²P]PP_i exchange data. In these experiments, DdaD, [³²P]PP_i, unlabeled ATP, and substrate were incubated and then ATP separated from PP_i by its specific adsorption to charcoal. The graph shows counts per minute arising from scintillography of the charcoal. (A) Adenylation activity with a variety of potential substrates. Reactions were quenched after 10 min. (B) Timecourse with NFmmDAPs, N_βEpSmDAPs, and dapdiamide A.

In the presence of both α-KG and DdaC, N_βFmmDAP-S-DdaD is converted to N_βEpSmDAP-S-DdaD. (A) In the presence of α-KG but the absence of DdaC, only the active site peptide from N_βFmmDAP-S-DdaD (blue) is observed. (B) In the presence of both α-KG and DdaC (prepared anaerobically), N_βFmmDAP-S-DdaD is converted to N_βEpSmDAP-S-DdaD and the active site peptide from N_βEpSmDAP-S-DdaD (orange) is observed. The theoretical mass shift of this transformation is 15.995 Da. (C) In the presence of α-KG and the absence of DdaC, only the small molecule Ppant ejection ion from N_βFmmDAP-S-DdaD (blue) is observed when the peptides are subjected to MS/MS using NSD. (D) In the presence of both α-KG and DdaC (prepared anaerobically), the Ppant ejection ions from both N_βFmmDAP-S-DdaD and N_βEpSmDAP-S-DdaD (orange) are observed when the peptides are subjected to MS/MS using NSD. For the predicted structures of the Ppant ejection ions, see Figure S6.

Proposed role of DdaC-F in the formation of N_βEpSmDAP-Val.