Identification and Localization of a Lipase-like Acyltransferase in Phenylpropanoid Metabolism of Tomato (Solanum lycopersicum)

Abstract

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...

Tomato GDSL1 Is Required for Cutin Deposition in the Fruit Cuticle

“…A biological role of GDSL-lipases in cutin deposition is in agreement with previous data showing that these proteins are localized to the apoplastic space of seedlings (Takahashi et al, 2010;Teutschbein et al, 2010) or to the epidermis of leaves and fruits (Reina et al, 2007;Matas et al, 2010;Yeats et al, 2010). Although the structure and composition of cutin were not characterized, the cosilencing of two Arabidopsis GDSL-lipases led to severe floral organ fusion and major changes in the architecture of epidermis cells .…”

“…In support of such an assertion, one can mention that the maize (Zea mays) acetylcholine esterase, Brassica napus sinapine esterase, and the chlorogenate:glucarate caffeoyltransferase of tomato seedlings share the same GDSL motif and the five conserved domains with GDSL1 (Sagane et al, 2005;Clauss et al, 2008;Teutschbein et al, 2010).…”

Requirement of Catalytic-Triad and Related Amino Acids for the Acyltransferase Activity ofTanacetum cinerariifoliumGDSL Lipase/Esterase TcGLIP for Ester-Bond Formation in Pyrethrin Biosynthesis

“…In respect of this enzyme, mutations of the catalytic-triad serine in Block I, as well as of the aspartate and histidine in Block V had only a small impact on the activity. 7) However, this may not be the case for the pyrethrin-forming acyltransferase activity of TcGLIP, since we have observed that the S40A mutation eliminated the activity. 4) We prepared in this present study MBP-fused and MBP-free TcGLIPs to examine the effects of MBP on the acyltransferase activity.…”

“…The D328A and H331A mutations, and also even the S27A mutation, only had a small impact on the glucarate caffeoyltransferase activity. 7) In the case of TcGLIP, however, mutations of the corresponding amino acids markedly reduced the pyrethrin-synthesizing acyltransferase activity. The results and our previous observation of the S40A mutation-induced complete loss of hydrolase activity 4) appear to suggest that the acyltransferase and hydrolase activities of TcGLIP shared a common mechanism.…”

“…1A), chrysanthemoyl CoA and (S)-pyrethrolone were added to a crude enzyme from the flower bud, resulting in the production of pyrethrin I. Purifying this enzyme and cloning the gene unexpectedly led to the discovery that the isolated enzyme showed similarities to GDSL lipases/esterases which have been named after the Gly-Asp-Ser-Leu motif. 4) This family consists of many members with such diverse functions as ceramidase, 5) sinapine esterase, 6) transesterification, 7) cutin synthesis 8) and cutinase activities 9) in plants. 10) We confirmed that the recombinant enzyme expressed in Escherichia coli as a fusion with maltosebinding protein (MBP) showed acyltransferase activity for pyrethrin synthesis.…”