Identification and localization of a lipase-like acyltransferase in phenylpropanoid metabolism of tomato (Solanum lycopersicum).
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.
Teutschbein
Jenny
J
Leibniz Institute of Plant Biochemistry, D06120 Halle (Saale), Germany.
Gross
Wiltrud
W
Nimtz
Manfred
M
Milkowski
Carsten
C
Hause
Bettina
B
Strack
Dieter
D
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002726:Chlorogenic Acid / Q000378:metabolism; D003373:Coumaric Acids / Q000378:metabolism; D018076:DNA, Complementary; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000502:physiology; D036226:Seedlings / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
D003373:Coumaric Acids; D018076:DNA, Complementary; D010940:Plant Proteins; D021382:Protein Sorting Signals; D002726:Chlorogenic Acid; D000217:Acyltransferases; D008049:Lipase