Biosynthesis Of Sinapoyl Esters

Sinapoyl esters are phenolic compounds found in members of the Brassicaceae, which includes the model plant Arabidopsis thaliana. The two major sinapoyl esters are sinapoyl malate (3.92) and sinapoyl choline (3.93), which accumulate in leaves and seeds, respectively. Sinapoylmalate plays a role in the protection against UV-radiation (Landry et al., 1995), whereas sinapoyl choline may be used as a storage form of choline in seeds (Shirley and Chapple, 2003). The precursor of these two esters is sinapate (3.35).

Sinapate is synthesized via the oxidation of sinapaldehyde (3.79) by an aldehyde dehydrogenase, as described in Section 13 of this chapter. Sinapaldehyde, in turn, is derived from the amino acid phenylalanine (3.27) via the general phenylpropanoid pathway (see Section 7), followed by a number of the hydroxylation and methylation reactions described in Section 10.

The first step in the biosynthesis of sinapoyl esters from sinapate (3.35) is glycosylation of the acid by the enzyme UDP-glucose:sinapic acid glucosyltransferase (SGT) to yield 1-O-sinapoylglucose (3.91). In leaves the enzyme sinapoylglucose:malate sinapoyltransferase (SMT) converts sinapoyl glucose (3.91) to sinapoyl malate (3.92). In seeds the enzyme sinapoylglucose:choline sinapoyltransferase (SCT) performs a similar reation to yield sinapoylcholine (3.93). This latter compound can be converted back to sinapate through the action of the enzyme sinapoylcholinesterase (SCE).

The elucidation of sinapoyl ester metabolism was aided by the availability of mutants. The sng1 (sinapoyl glucose accumulator 1) mutant of Arabidopsis had been identified based on a mutant screen for alterations in the composition of fluorescent compounds in the leaves. The screen was performed by thin layer chromatography and revealed that the leaves of the sng1 mutant contained less sinapoylmalate and instead accumulated the precursor sinapoyl glucose (Lorenzen et al. 1996).

The Arabidopsis SNG1 gene was cloned using a map-based cloning approach. The gene was shown to encode a serine carboxypeptidase-like (SCPL) protein (Lehfeldt et al., 2000). This class of enzymes has been shown to be involved in protein degradation, whereby the peptide bond between the penultimate and C-terminal amino acid residues of the protein or peptide substrates is cleaved. Expression of the Arabidopsis SNG1 protein in E. coli demonstrated SMT activity, and hence that this enzyme catalyzes a transesterification reaction, as opposed to the hydrolytic reaction typical for serine carboxypeptidases.

The SCT gene was also cloned based on the availability of a mutant. The Arabidopsis sng2 mutant (sinapoylglucose accumulator 2) was identified in a mutant screen whereby seed extracts were analyzed with thin layer chromatography for the accumulation of sinapoylglucose (Shirley et al., 2001). The Arabidopsis SNG2 gene was cloned via a combination of map-based cloning and a candidate gene approach. The candidate gene in this case was an SCPL-gene that had been identified during the sequencing of the

Arabidopsis genome, and that mapped to the same chromosome as the SNG2 locus. This candidate gene indeed turned out to be the SNG2 gene, as was demonstrated via complementation of the mutant. Activity towards sinapoylglucose was shown by expressing the SNG2 cDNA in E. coli More detailed kinetic studies of recombinant SCT isolated from yeast are described by Shirley and Chapple (2003).

UDP-Glc UDP

malate

choline

d choline

O^l glucose c

H3C-N-CH3

d choline

O^l glucose

H3C-N-CH3

glucose

malate glucose

Figure 3-13. Sinapoyl ester metabolism catalyzed by the enzymes (a) UDP-glucose:sinapic acid glucosyltransferase (SGT), (b) sinapoylglucose:malate sinapoyltransferase (SMT), (c) sinapoylglucose:choline sinapoyltransferase (SCT), and (d) sinapoylcholinesterase (SCE).

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