Extended Data Fig. 8: Evaluation of CybE substrate preference by exchange of the aliphatic (β) building block.

Individual assays with At4CL1, CybE, and CybF with the alternative aliphatic building blocks 3-methyl-2-oxopentanoic acid (derived from isoleucine) and 4-methyl-2-oxopentanoic acid were conducted in comparison to the cyanobacterin precursor 3-methyl-2-oxobutanoic acid. Strong preference of CybF towards 3-methyl-2-oxopentanoic acid leading to 8 is indicated by significantly higher amounts of product 8 when compared to 24 formation. In addition, competition assays with both alternative precursors lead to exclusive formation of 8. Depicted assays with substrates: 3-methyl-2-oxobutanoic acid (i); 3-methyl-2-oxopentanoic acid (ii); 4-methyl-2-oxopentanoic acid (iii). competition assay simultaneously using 500 µM 3-methyl-2-oxopentanoic acid and 500 µM with 4-methyl-2-oxopentanoic acid (iv). To exclude failed detection of S1 in assay (iv) due to potentially insufficient chromatographic separation of 8 and S1, samples (iii) and (iv) were mixed in a 1:1 ratio and reanalyzed by HPLC (v). As can be seen (box) there is separation of 8 and 24 and hence the absence of the 24 peak in chromatogram (iv) is due to substrate preference of CybE for 3-methyl-2-oxopentanoic acid over with 4-methyl-2-oxopentanoic acid. Structures of all compounds are provided at the top of the figure.