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. Author manuscript; available in PMC: 2023 Aug 30.
Published in final edited form as: J Am Chem Soc. 2022 Nov 17;144(47):21628–21639. doi: 10.1021/jacs.2c09004

Figure 2: Posttranslational modifications to the amycolimiditide precursor.

Figure 2:

A: Co-expression of amycolimiditide precursor AmdA with ATP-grasp enzyme AmdB leads to 4 dehydrations (4DH) of AmdA while coexpression of AmdA with AmdB and PIMT homolog AmdM leads to 5 dehydrations (5DH) of AmdA. In all cases, AmdB is present as a SUMO fusion protein. Dotted guide lines correspond to the calculated average mass of a given species. B: Time course of aspartimidylation of mAmdAB by AmdM in vitro. Methylated mAmdAB (4DHMe) is present at early times but is converted to the aspartimide within 3 h. C: EIC chromatograms of amycolimiditide (top), its hydrolysis products (middle) and pre-amycolimiditide (bottom) show that amycolimiditide hydrolyzes into two distinct products. D: Time courses of methylation and aspartimidylation of pre-amycolimiditide (containing Asp) and iso pre-amycolimiditide (containing isoAsp). Methylation of pre-amycolimiditide proceeds faster than methylation of iso pre-amycolimiditide, but aspartimide formation is faster for iso pre-amycolimiditide than for pre-amycolimiditide. Experiments were run in triplicate with all data points shown. Error bars represent the standard deviation; some error bars are so small that they are not visible.