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(Last
modification:05. February 2010)
Results obtained in vitro with the recombinant
type III PKS from Hydrangea macrophylla
Note: the results with these two substrates are
different, see below. The enzyme synthesized a stilbenecarboxylate with
dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA: see the publication for a
discussion of the likely reasons: Eckermann et al. 2003.
Or go directly to the discussion of the reaction
mechanisms of STCS !
4-Coumaroyl-CoA
This is the substrate predicted for the biosynthesis of hydrangeic acid. The results of the in vitro assays are shown below.
The results clearly showed that the protein was not a chalcone synthase or stilbene synthase (with this substrate: a resveratrol synthase), but the expected stilbenecarboxylic acid was also not detected. We found instead CTAL and bisnoryangonin, in a ratio of 6 : 1 (i.e. the enzyme preferentially carried out three condensations). As discussed elsewhere in this website, these two are
byproducts typically found with CHS and STS under conditions in vitro that are not optimal or otherwise lack a critical component of the biological system.
Important
While our work was in progress, cDNAs for a CHS and a CHS-related protein from another Hydrangea variety, Hydrangea macrophylla var. thunbergii, were reported.
This is a different Hydrangea variety: the spectrum of the natural products overlaps with that of the Garden Hortensia, but some substances were found only in the thunbergii variety, while others were described only from the Garden Hortensia, and therefore the results of that study will be discussed separately:
More...
Dihydro-4-Coumaroyl-CoA
Evidence that this type III PKS is indeed a stilbenecarboxylate synthase
This is the substrate predicted for the biosynthesis of lunularic acid, a natural product also known from H. macrophylla. The results of the in vitro assays are shown below.
The lack of the products expected from the ring-folding typical for chalcone synthase (CHS) and stilbene synthase (STS) confirmed that the protein had not one of those enzyme activities. A clear difference to the results with 4-coumaroyl-CoA was that a third product was found (in addition to the pyrones from two and three condensations), and this could be identified as
5-hydroxylunularic acid, the product expected from a stilbenecarboxylate synthase (STCS) activity. Actually, this product represented 40-45 % of the product mixture, indicating that it was not just a minor result of the reaction.
These results demonstrated that the CHS-related protein from Hydrangea macrophylla functioned as stilbenecarboxylate (STCS) with this substrate.
The same results were obtained with two CHS-related
type III PKS from Marchantia polymorpha:
More...
The publication
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Eckermann, C., Schröder, G., Eckermann, S., Strack, D., Schmidt, J., Schneider, B., and Schröder, J.: Stilbenecarboxylate biosynthesis: a new function in the family of chalcone synthase-related proteins. Phytochemistry 62, 271-286 (2003).
Chalcone (CHS), stilbene (STS) synthases, and related proteins are key enzymes in the biosynthesis of many secondary plant products. Precursor feeding studies and mechanistic rationalization suggest that stilbenecarboxylates might also be synthesized by plant type III polyketide synthases; however, the enzyme activity leading to retention of the carboxyl moiety in a stilbene backbone has not yet been demonstrated. Hydrangea macrophylla L. (Garden Hortensia) contains stilbenecarboxylates (hydrangeic acid and lunularic acid) that are derived from 4-coumaroyl and dihydro-4-coumaroyl starter residues, respectively. We used homology-based techniques to clone CHS-related sequences, and the enzyme functions were investigated with recombinant proteins. Sequences for two proteins were obtained. One was identified as CHS. The other shared 65-70% identity with CHSs and other family members. The purified recombinant protein had stilbenecarboxylate synthase (STCS) activity with dihydro-4-coumaroyl-CoA, but not with 4-coumaroyl-CoA or other substrates. We propose that the enzyme is involved in the biosynthesis of lunularic acid. It is the first example of a STS-type reaction that does not lose the terminal carboxyl group during the ring folding to the end product. Comparisons with CHS, STS, and a pyrone synthase showed that it is the only enzyme exerting a tight control over decarboxylation reactions. The protein contains unusual residues in positions highly conserved in other CHS-related proteins, and mutagenesis studies suggest that they are important for the structure or/and the catalytic activity. The formation of the natural products in vivo requires a reducing step, and we discuss the possibility that the absence of a reductase in the in vitro reactions may be responsible for the failure to obtain stilbenecarboxylates from substrates like 4-coumaroyl-CoA.
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