We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.Plant metabolism is characterized by the formation of a vast number of secondary compounds, brought about by gene families coding for enzymes that modify various phenolic, terpenoid, alkaloid, or polyketide skeletons by oxidation and reduction as well as by methylation, glycosylation, prenylation, and acylation. Most of the phenolic structures in plants are synthesized via the shikimate/hydroxycinnamate pathway, which feeds into different types of hydroxycinnamate (HCA) 4 sidechain reactions (1). Among them are extensions with formation of additional ring systems (e.g. flavonoids or stilbenes), degradation (e.g. hydroxybenzoates), reduction (e.g. hydroxycinnamyl alcohols feeding into lignin biosynthesis), oxidation and lactonization (e.g. coumarins), and conjugation with a wide range of different primary and secondary compounds to form esters or amides. Syntheses of HCA conjugates are catalyzed by hydroxycinnamoyltransferases that play a decisive role in catalyzing the formation of complex patterns of HCA esters (2). Such a pattern, for example, was recently identified from Brassica napus seeds and exhibited a mixture of sinapate esters containing choline, malate, mono-and disaccharides, as well as flavonoid glycosides and an unusual cyclic spermidine amide (3).Tomato (Solanum lycopersicum) leaves accumulate caf...

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