Enzymes of plant gallotannin metabolism
Final Report Abstract
Work of this funding period was focused on the characterization of shikimate dehydrogenases (SDH) and the identification of a galloyltransferase (GALT) from Quercus robur. Three cDNA candidates for SDH from Q. robur, QrSDH1, QrSDH2 and QrSDH3, were expressed in E. coli and in Nicotiana benthamiana. The recombinant enzymes were tested for SDH activity and for the ability to catalyze the NADP+-dependent oxidation of 3-dehydroshikimic acid to produce gallic acid. In the E. coli expression strain M15 [pREP4], the production of soluble QrSDH failed. Therefore, we developed a tailored bacterial expression system for QrSDH enzymes exploiting genetic complementation of the aroE mutation in E. coli AB2834. In vitro assays with the recombinant QrSDH enzymes produced in E. coli confirmed the SDH activity for QrSDH1-3, but gallic acid formation was not observed. The same outcome was achieved with recombinant QrSDH1-3 enzymes produced in N. benthamiana. For the identification of a galloyltransferase, a protein purification protocol was developed to enrich the enzyme converting 1-O-galloylglucose to 1,6-di-O-galloylglucose from oak leaves. MS-based peptide analysis of the active protein fraction revealed significantly increased amounts of a β-glucosidase-like enzyme (QrGALT). Two candidate cDNAs, QrGALT1 and QrGALT2, were cloned and expressed in leaves of Nicotiana benthamiana. In vitro assays confirmed β-glucosidase activity but failed in the detection of galloyltransferase activity. To increase the number of galloyltransferase candidates, we cloned a cDNA from leaves of Q. robur encoding a putative serine carboxypeptidase-like (SCPL) acyltransferase (QrSCPL1), based on reports on a galloyltransferase activity from tea plant. Heterologous expression in leaves of N. benthamiana followed by in vitro assay of enzyme activity did not produce evidence of a galloyltransferase activity mediated by QrSCPL1. During the actual funding period we established a cooperation with Dr. Ko Tahara from the Forestry and Forest Products Research Institute, Tsukuba, Japan. As a result of the collaborative work, two ester-forming gallic acid glucosyltransferases and one SDH could be identified from the economically important tree Eucalyptus camaldulensis.
