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Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.

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Abstract

Five glucosyltransferases were cloned by RT-PCR amplification using total RNA from Hieracium pilosella L. (Asteraceae) inflorescences as template. Expression was accomplished in Escherichia coli, and three of the HIS-tagged enzymes, UGT90A7, UGT95A1, and UGT72B11 were partially purified and functionally characterised as UDP-glucose:flavonoid O-glucosyltransferases. Both UGT90A7 and UGT95A1 preferred luteolin as substrate, but possessed different regiospecificity profiles. UGT95A1 established a new subgroup within the UGT family showing high regiospecificity towards the C-3′ hydroxyl group of luteolin, while UGT90A7 primarily yielded the 4′-O-glucoside, but concomitantly catalysed also the formation of the 7-O-glucoside, which could account for this flavones glucoside in H. pilosella flower heads. Semi quantitative expression profiles revealed that UGT95A1 was expressed at all stages of inflorescence development as well as in leaf and stem tissue, whereas UGT90A7 transcript abundance was nearly limited to flower tissue and started to develop with the pigmentation of closed buds. Other than these enzymes, UGT72B11 showed rather broad substrate acceptance, with highest activity towards flavones and flavonols which have not been reported from H. pilosella. As umbelliferone was also readily accepted, this enzyme could be involved in the glucosylation of coumarins and other metabolites.

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Abbreviations

GT:

Glycosyltransferases

UGT:

UDP-Glycosyltransferases

PSPG:

Plant secondary product glycosyltransferases

RACE:

Rapid amplification of cDNA ends

HSCCC:

High-speed counter current chromatography

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft and Dr. Sofia Moco is financed by the EU project “META-PHOR” (FOOD-CT-2006-036220).

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Authors and Affiliations

  1. Institut Für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17 A, 35037, Marburg, Germany

    Simone Witte, Ulrich Matern & Stefan Martens

  2. Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA, Wageningen, The Netherlands

    Sofia Moco & Jacques Vervoort

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  1. Simone Witte
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  2. Sofia Moco
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  3. Jacques Vervoort
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  4. Ulrich Matern
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Correspondence to Stefan Martens.

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Chemical structures of flavones occurring in H. pilosella (TIFF 316 kb)

425_2009_902_MOESM2_ESM.tif

Alignment of H. pilosella UGT polypeptides using the ClustalW program. The conserved PSPG motif is shown in bold letters. UGT72B11 (GenBank accession number EU561016), UGT88A8 (EU561017), UG88A9 (EU561018), UGT90A7 (EU561019), UGT95A1 (EU561020) (TIFF 65 kb)

Abbreviations and GenBank accession numbers (DOC 103 kb)

Supplementary Table 1S (DOC 132 kb)

Supplementary Table 2S (DOC 145 kb)

Supplementary Table 3S (DOC 134 kb)

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Witte, S., Moco, S., Vervoort, J. et al. Recombinant expression and functional characterisation of regiospecific flavonoid glucosyltransferases from Hieracium pilosella L.. Planta 229, 1135–1146 (2009). https://doi.org/10.1007/s00425-009-0902-x

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