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cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis

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Abstract

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.

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Acknowledgements

We are thankful for Dr. Stefan Martens’ and Dr. Anna Berim’s help with the yeast expression system in our laboratory.

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Authors and Affiliations

  1. Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstr. 17A, 35037, Marburg, Germany

    David Eberle & Maike Petersen

  2. Départment Résponses Métaboliques, Institut de Biologie Moléculaire des Plantes, CNRS-UPR2357, Université Louis Pasteur, 28 rue Goethe, 67000, Strasbourg, France

    Pascaline Ullmann & Danièle Werck-Reichhart

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  1. David Eberle
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  2. Pascaline Ullmann
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Correspondence to Maike Petersen.

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Eberle, D., Ullmann, P., Werck-Reichhart, D. et al. cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from Coleus blumei involved in rosmarinic acid biosynthesis. Plant Mol Biol 69, 239–253 (2009). https://doi.org/10.1007/s11103-008-9420-7

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