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iScience
2024 Aug 20;279:110834. doi: 10.1016/j.isci.2024.110834.
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Molecular basis of pigment structural diversity in echinoderms.
Li F
,
Lin Z
,
Schmidt EW
.
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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.
Figure 1. Polyketides and polyketide synthases (PKSs) from echinoderms(A) SpPks1 homologs found in echinoderms. The phylogenetic tree was made using ketosynthase (KS) domain sequence alignments and reflects the preservation of essential SpPks1 homologs over >500 million years of evolution.(B) Aromatic pigments previously reported from crinoids (Crinoidea) Anneissia japonica, which encodes CrPKS, and Alloeocomatella polycladia.(C) Examples of other echinoderm polyketide structural families from sea stars (Asteroidea), sea cucumbers (Holothuroidea), and brittle stars (Ophiuroidea).(D) Biosynthesis of ATHN (10) by the enzyme SpPks1 from sea urchins (Echinoidea). Further echinoderm-derived polyketides are shown in Figure S1.
Figure 2. Architecture of CrPKS(A) CrPKS and SpPks1 have identical domain orders. Domains include: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; cMT, a pseudo-methyltransferase structural domain with no catalytic activity; ER, enoylreductase; KR, ketoreductase; ACP, acyl carrier protein.(B) CrPKS KR domain is likely inactive, consistent with the synthesis of aromatic polyketides. Essential residue Y2209 is thought to be required for the activity of KR domains. It is present in a wide range of reducing PKSs and FASs from fungi and animals, but absent in the KR domain of SpPks1 and CrPKS, indicating a potentially inactive ketoreductase domain required for aromatic polyketide biosynthesis. Numbering according to CrPKS. mFAS: mammalian FAS, EcPKS1 and EcPKS2: biochemically characterized HRPKSs from mollusk Elysia chlorotica; LovB: HRPKS from fungus Aspergillus terreus.
Figure 3. CrPKS synthesizes 14-carbon pigment 13(A) HPLC (254 nm), extracted chromatograms.(B) UV chromatograms showing fungal products (magenta) and CrPKS reaction products (blue).(C and D) Mass spectra (negative mode) of 13 when CrPKS was incubated with (C) malonyl-CoA or (D) 13C malonyl-CoA. Purple dots indicate the incorporation of 13C. Purified protein is shown in Figure S2, while additional related data are shown in Figures S3–S6. Data showing the MS- and NMR-based identification of compound 13 can be found in Figures S7–S10. Figures S11 and S12 show that NADPH is not required and that either acetyl- or malonyl-CoA can be used as starter units.
Figure 4. CrPKS synthesizes pigments with saturated side chains(A–D) Mass spectral chromatograms (negative mode) of the reaction mixture containing CrPKS and both malonyl- and butyrl-CoA, filtered for (A) m/z 275.0651 corresponding to compound 17 and (B) m/z 261.0768 corresponding to 18. Enzymatic reactions were also performed with 13C-malonate, and the labeled (top) and unlabeled (bottom) mass spectra were compared for (C) compound 17 and (D) compound 18. The addition of 5 units of malonate is reflected in the mass shift and further reinforced in Figures S13 and S4. Purple dots indicate the incorporation of 13C. Further MS data supporting this figure can be found in Figures S13–S17.
Figure 5. A KS domain mutation that controls chain length and starter unit selectivity(A) Conserved residues in SpPks1 and CrPKS.(B) Purified CrPKS-GMMD synthesizes more compound 10 than does wild-type CrPKS. The y axis is the ratio of [12-carbon products]/([12-carbon products] + [14-carbon products]) for CrPKS (magenta) and CrPKS-GMMD (green), while the x axis indicates the concentration of malonyl-CoA in mM. See Figures S19–S21 for details. Each point was replicated three times, with error bars representing the standard deviation.(C) In yeast cell pellets, CrPKS-GMMD synthesizes more compound 10 than does CrPKS (see Figures S23 and S24 for additional details).(D) In yeast cell pellets, SpPks1-GLMD synthesizes a small amount of compound 13 (see Figure S22).(E and F) Michaelis-Menten kinetics for CrPKS and (F) for CrPKS-GMMD. Curves are shown for different concentrations of malonyl-CoA (blue) or butyryl-CoA (magenta) in the presence of 2 mM malonyl-CoA. Each point was replicated three times, with error bars indicating standard deviation.(G an H) The corresponding kinetic constants generated from the curves shown in panels (E) and (F). Time course experiments underlying kinetics are in Figure S25.(I and J) AlphaFold models of the KS domains of (I) CrPKS and (J) SpPks1. The models show the distance between active-site cysteine and the mutated residue. Further models are shown in Figure S18. Primers used to generate mutants are found in Table S1.
Figure 6. Plausible biosynthetic routes to pigments isolated from CrinoideaThe top box indicates potential pathways surmised based on experiments with CrPKS, while the bottom box focuses on reactions catalyzed by SpPks1. Also, note that 13 is spontaneously hydrolyzed to 14, a feature that was used in the NMR characterization of products. Potential anthraquinone biosynthesis is shown in Figure S26. The biosynthetic mechanisms leading to compounds 10 and 13 in echinoderms and fungi are shown in Figure S27.
