Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Two novel Physcomitrella patens fatty acid elongases (ELOs): identification and functional characterization Pradinunt Eiamsa-ard & Akkharawit Kanjana-Opas & Edgar B. Cahoon & Pichit Chodok & Sireewan Kaewsuwan
Received: 21 July 2012 / Revised: 25 October 2012 / Accepted: 25 October 2012 / Published online: 9 November 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract The lower plant Physcomitrella patens synthesizes several long-chain polyunsaturated fatty acids (LCPUFAs) by a series of desaturation and elongation reactions. In the present study, the full-length cDNAs for two novel fatty acid elongases designated PpELO1 and PpELO2 were isolated from P. patens using a PCR-based cloning strategy. These cDNAs encoding proteins of 335 and 280 amino acids with predicted molecular masses of 38.7 and 32.9 kDa, respectively, are predicted to contain seven transmembrane domains with a possible localization in the subcellular endoplasmic reticulum. Sequence comparisons and phylogenetic analysis revealed that they are closely related to other LC-PUFA elongases of the lower eukaryotes such as the Δ5- and Δ6-elongases of Marchantia polymorpha as well as the Δ6-elongase of P. patens. Heterologous expression of the PpELO1 in Saccharomyces cerevisiae led to the elongation of Δ9-, Δ6-C18, and Δ5-C20 LC-PUFAs, whereas
only Δ9- and Δ6-C18 LC-PUFA substrates were used by PpELO2. Chimeric proteins were constructed to identify the amino acid regions most likely to be involved in the determination of the fatty acid substrate specificity. The expression of eight chimeric proteins in yeast revealed that substitution of the C-terminal 50 amino acids from PpELO1 into PpELO2 resulted in a high specificity for C20 fatty acid substrates. As a result, we suggest that the C-terminal region of PpELO1 is sufficient for C20 substrate elongation. Overall, these results provide important insights into the structural basis for substrate specificity of PUFAgenerating ELO enzymes.
Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4556-4) contains supplementary material, which is available to authorized users.
Introduction
P. Eiamsa-ard : A. Kanjana-Opas Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112, Thailand E. B. Cahoon Center for Plant Science Innovation and Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE 68588, USA P. Chodok : S. Kaewsuwan (*) Marine Natural Products Research Unit (MNP), Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Songkhla 90112, Thailand e-mail: [email protected] S. Kaewsuwan e-mail: [email protected]
Keywords Physcomitrella patens . Polyunsaturated fatty acid (LC-PUFA) . Fatty acid elongase (ELO) . Chimeric protein
Long-chain polyunsaturated fatty acids (LC-PUFAs) are defined as fatty acid chains comprised of 20 or more carbons with a terminal carboxyl group and hav
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