PMC

Kinetic analysis of DCS. The rate-feruloyl-CoA concentration profile (A), Eadie-Hofstee plot (B), and the rate-malonyl-CoA concentration profile (C) are shown. The data, obtained from three independent experiments, suggest that DCS is an allosteric enzyme.

Kinetic analysis of CURS. The rate-feruloyl-CoA concentration profile (A) for cinnamoylferuloylmethane (6e) synthesis from feruloyl-CoA (3a) and cinnamoyldiketide-NAC (4b) are shown. Similarly, the rate-p-coumaroyl-CoA concentration profile (B) for cinnamoyl-p-coumaroylmethane (5e) synthesis from p-coumaroyl-CoA (2a) and cinnamoyldiketide-NAC (4b) are shown. The data were obtained from three independent experiments.

Analysis of the ethyl acetate extracts of reactions containing DCS. 4-Hydroxybenzalacetone (2d) was not detected in the extract of the reaction containing p-coumaroyl-CoA (2a), malonyl-CoA, and DCS (A) but appeared in the extract after alkaline hydrolysis (C). Dehydrozingerone (3d) in a low amount was detected in the extract of the reaction containing feruloyl-CoA (3a), malonyl-CoA, and DCS (B). The yield of dehydrozingerone (3d) increased after alkaline hydrolysis of the reaction mixture containing feruloyl-CoA (3d), malonyl-CoA, and DCS (D). 4-Hydroxybenzalacetone (E) and dehydrozingerone (F) were identified by comparing their retention times, MS, MS/MS, and UV spectra with those of the authentic samples.

HPLC analysis of the reaction products of DCS. Incubation of DCS in the presence of feruloyl-CoA (3a) and malonyl-CoA yielded feruloyldiketide-CoA (3b) (A), whereas the reaction with boiled DCS, as a negative control, yielded no products (B). The UV spectra of feruloyldiketide-CoA (3b) and feruloyl-CoA (3a) are shown in the insets in A and B, respectively. The comparison of MS/MS spectra of feruloyldiketide-CoA (3b) (C) and feruloyl-CoA (3a) (D) showed that the product was feruloyldiketide-CoA. The parent ion peaks of feruloyldiketide-CoA (3b) and feruloyl-CoA (3a) are shown in the insets in C and D, respectively. The predicted fragmentation patterns of feruloyldiketide-CoA (E) and feruloyl-CoA (F) are shown.

HPLC analysis of the reaction products of DCS/CURS co-incubation. The front chromatograms show control reactions containing only CURS, and the back chromatograms indicate the reactions containing both DCS and CURS. Co-incubation of DCS and CURS in the presence of cinnamoyl-CoA (1a) and malonyl-CoA yielded a trace amount of dicinnamoylmethane (1e), whereas the control reaction containing only CURS did not (A). Co-incubation of DCS/CURS in the presence of p-coumaroyl-CoA (2a) (B) or feruloyl-CoA (3a) (C), in addition to malonyl-CoA, yielded a larger amount of bisdemethoxycurcumin (2e) or curcumin (3e), respectively, than the control incubation of only CURS (B and C).

Curcuminoid biosynthesis in C. longa, as proposed by the present study (A) and the enzyme activities of various type III PKSs, including DCS and CURS (B). A, the starter substrates, cinnamoyl-CoA (1a), p-coumaroyl-CoA (2a), or feruloyl-CoA (3a), are synthesized from phenylalanine by the activity of phenylalanine ammonia-lyase (PAL), 4-coumarate:CoA ligase (4CL), cinnamate-4-hydroxylase (C4H), hydroxycinnamoyl transferase (HCT), cinnamate-3-hydroxylase (C3H), and O-methyltransferase (OMT). DCS catalyzes formation of diketide-CoAs by condensing p-coumaroyl-CoA and feruloyl-CoA with malonyl-CoA. CURS catalyzes formation of curcuminoids by condensing diketide-CoAs with starter substrates. B, the reactions catalyzed by DCS, CURS, benzalacetone synthase (BAS) from Rheum palmatum, CUS from O. sativa, and CHS are shown.

HPLC analysis of the reaction products of CURS. The scheme of the CURS reaction starting with CoA esters and cinnamoyl-diketide-NAC (4b) (A) is illustrated. In chromatograms B–D, the front chromatograms show the CURS reactions beginning with a starter substrate and malonyl-CoA and the back chromatograms show the CURS reaction beginning with a starter substrate and cinnamoyl-diketide-NAC (4b). Incubation of CURS in the presence of p-coumaroyl-CoA (2a) (C, front) and feruloyl-CoA (3a) (D, front), in addition to malonyl-CoA, yielded bisdemethoxycurcumin (2e) and curcumin (3e), respectively. No products were detected in the reactions starting with cinnamoyl-CoA (1a) and malonyl-CoA (B, front). When cinnamoyl-CoA (1a) (B, back), p-coumaroyl-CoA (2a) (C, back), or feruloyl-CoA (3a) (D, back) were added to a reaction containing cinnamoyl-diketide-NAC (4b), dicinnamoylmethane (1e), cinnamoyl-p-coumaroylmethane (5e), or cinnamoylferuloylmethane (6e), respectively, were produced. The dicinnamoylmethane (E, back), bisdemethoxycurcumin (F, front), cinnamoyl-p-coumaroylmethane (F, back), curcumin (G, front), and cinnamoylferuloylmethane (G, back) were identified by comparing their retention times, MS, MS/MS, and UV spectra with those of the authentic or synthetic samples.