Fig. 5: Characterization of the barley diketone metabolism polyketide synthase (HvDMP) catalyzing the condensation of 3-ketoacids and acyl-CoAs into β-diketones.

GC-MS analysis of in vivo and in vitro assays feeding acyl-CoA and/or ketoacid substrates to HvDMP. Chromatograms of fragment m/z 100 selectively reporting β-diketones are shown on the left and mass spectra of key products on the right. A Chromatogram of lipids from yeast expressing HvDMP and supplemented with C₁₆ 3-ketoacid (n = 11, 13). B Mass spectra of C₃₁ 14,16-diketone peaks in (A, upper panel) and (C, lower panel). C Chromatogram of lipids from HvDMP in vitro assay with C₁₆ 3-ketoacid and C₁₆ acyl-CoA. Boiled recombinant enzyme incubated with C₁₆ 3-ketoacid and C₁₆ acyl-CoA served as control. D Chromatograms of characteristic fragments of lipids from yeast expressing HvDMP and supplemented with C₁₆ 3-ketoacid and per-deuterated fatty acid C₁₅D₃₁COOH, selectively reporting undeuterated (dashed lines) and deuterated β-diketones (solid lines). E Mass spectrum of the D₃₁-labeled C₃₁ 14,16-diketone in (D), showing α-fragment, McLafferty rearrangement and water/acetone loss ions diagnostic for the C₁₄ acyl moiety of C₃₁ 14,16-diketone. Analogous fragments of the other hydrocarbon tail were shifted by 31 amu relative to the unlabeled C₃₁ 14,16-diketone, as were the molecular ion and the water loss ion [M-18]⁺. A cluster of ions around m/z 103 is consistent with double-McLafferty rearrangement on both sides of the β-diketo group, a double-deuterated α-methylene and γ-deuterium transfer. Together, the GC and MS characteristics unambiguously identified this compound as C₃₁ 14,16-diketone with C₁₃H₂₇ and C₁₅D₃₁ hydrocarbon tails. F Chromatogram of lipids from yeast expressing HvDMP and supplemented with even-numbered 3-ketoacid (C₁₆) and odd-numbered fatty acid (C₁₅). G Mass spectra of C₃₀ 14,16-diketone in (F, upper panel) and (H, lower panel). H Chromatogram of lipids from yeast expressing HvDMP and supplemented with odd-numbered 3-ketoacid (C₁₇) and even-numbered fatty acid (C₁₄). Arabidopsis LACS1 was expressed in all the yeast assays to enhance exogenous substrate uptake⁶¹.