Figure 5.
In vitro reconstitution of the PKS and the glycosyltransferase proteins involved in pactamycin biosynthesis. a. Enzymatic reactions from 3ABA to GlcNac-3ABA-PtmI and to GlcNAc-β-ketoacyl-PtmI; b. Deconvoluted mass spectrum of a reaction mixture containing PtmS, PtmI, and PtmJ; c. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and PtmJ; d. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and PtmJ using 3ABA-2,4,5,6-d4 as substrate; e. Deconvoluted mass spectrum of a reaction mixture with PtmS, PtmI, PtmK, and boiled PtmJ. Magenta circle, holo-PtmI; blue circle, acetyl-PtmI; black circle, malonyl-PtmI; red diamond, 3ABA-PtmI; purple triangle, 3AP-3OP-PtmI; orange star, GlcNAc-3ABA-PtmI; yellow star, GlcNAc-3AP-3OP-PtmI. The gluconoylated proteins are labeled with “Gluc”. Deuterated species are labeled with d4. Experiments 5b-e were performed independently at least three times with similar results.