Molecular basis of pigment structural diversity in echinoderms

Summary The varied pigments found in animals play both ecological and physiological roles. Virtually all echinoderms contain putative pigment biosynthetic enzymes, the polyketide synthases (PKSs). Among these, crinoids have complex pigments found both today and in ancient fossils. Here, we characterize a key pigment biosynthetic enzyme, CrPKS from the crinoid Anneissia japonica. We show that CrPKS produces 14-carbon aromatic pigment precursors. Despite making a compound previously found in fungi, the crinoid enzyme operates by different biochemical principles, helping to explain the diverse animal PKSs found throughout the metazoan (animal) kingdom. Unlike SpPks1 from sea urchins that had strict starter unit selectivity, CrPKS also incorporated starter units butyryl- or ethylmalonyl-CoA to synthesize a crinoid pigment precursor with a saturated side chain. By performing biochemical experiments, we show how changes in the echinoderm pigment biosynthetic enzymes unveil the vast variety of colors found in animals today.


Supporting Table and Figures.
Figure S1: Pigments previously reported from Crinoidea, related to Figure 1                         Table S1: Primers used in this study, related to Figure 5.

Figure S2 :
Figure S1: Pigments previously reported from Crinoidea, related to Figure 1 and the Introduction.

Figure S3 :
Figure S3: LC-MS chromatograms of CrPKS enzyme assay with malonyl-CoA, related to Figure 3. A) Base-peak intensity ion chromatogram of assay (A2), compared to boiled enzyme control (A1); B) Peaks shown with blue arrows had an observed m/z at 275.0586 and 289.0378, corresponding to known compounds 13 and 16, respectively.

Figure S8 :
Figure S8: 1 H NMR of YWA2 (14), related to Figure 3 and STAR Methods.Top: NMR spectrum.Bottom: Conversion of 13 to 14 was accomplished synthetically in acidic conditions.

Figure S11 :
Figure S11: NADPH does not alter CrPKS product profile, related to Figure 3 and STAR Methods.CrPKS incubated with 2 mM malonyl-CoA A) without NADPH; B) with NADPH.

Figure S13 :
Figure S13: LC-MS chromatograms of CrPKS enzyme assay with malonyl-CoA and butyryl-CoA, related to Figure 4 and STAR Methods.A) New peaks were detected when CrPKS was incubated with a mixture of malonyl-CoA and butyryl-CoA, seen at m/z 275.0651.B) Molecular ions of the new peak, showing results when CrPKS was incubated with butyryl-CoA and either malonylor 13 C3-malonyl-CoA.Five units of malonate were incorporated in the new products.

Figure S17 :
Figure S17: Butyryl-CoA is a preferable starter unit in comparison to ethylmalonyl-CoA, related to Figure 4 and STAR Methods.CrPKS incubated with malonyl-CoA and A) ethylmalony-CoA; B) butyryl-CoA.The number above the peak indicates the area under curve. .

Figure S20 :
Figure S20: CrPKS-GMMD uses both butyryl-CoA and ethymalonyl-CoA starter units to synthesize compound 18, related to Figure 5 and STAR Methods.Extracted ion chromatograms of compound 18 from CrPKS incubated with malonyl-CoA and A) butyryl-CoA, B) ethylmalonyl-CoA.A smaller amount of compound 18 was synthesized with ethylmalonyl-CoA than with butyryl-CoA.

Figure S25 :
Figure S25: Time course experiment used to determine conditions for kinetics, related to Figure 5 and STAR Methods.A) CrPKS and B) CrPKS-GMMD reaction with butyryl-CoA (2 mM) and malonyl-CoA (2 mM), showing area under the curve (AUC) for compound 13.