Abstract
The search of the Phaeodactylum tricornutum genome database revealed the existence of six genes potentially encoding lysophospholipid acyltransferases. One of these genes, Phatr3_J20460, after introduction to yeast ale1 mutant disrupted in the LPCAT gene, produced a very active acyl-CoA:lysophosphatidylcholine (LPCAT) enzyme. Using in vitro assays applying different radioactive and non-radioactive substrates and microsomal fractions from such yeast, we have characterized the biochemical properties and substrate specificities of this PtLPCAT1. We have found that the substrate specificity of this enzyme indicates that it can completely supply phosphatidylcholine (PC) with all fatty acids connected with a biosynthetic pathway of very long-chain polyunsaturated fatty acids (VLC-PUFAs) used further for the desaturation process. Additionally, we have shown that biochemical properties of the PtLPCAT1 in comparison to plant LPCATs are in some cases similar (such as the dependency of its activity on pH value), differ moderately (such as in response to temperature changes), or express completely different properties (such as in reaction to calcium and magnesium ions or toward some acyl-CoA with 20C polyunsaturated fatty acids). Moreover, the obtained results suggest that cloned “Phatr3_J20460” gene can be useful in oilseeds plant engineering toward efficient production of VLC-PUFA as LPCAT it encodes can (contrary to plant LPCATs) introduce 20:4-CoA (n-3) to PC for further desaturation to 20:5 (EPA, eicosapentaenoic acid).
Highlights
Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdańsk, 80-307 Gdansk, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, China; Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan 430062, China
To identify the putative lysophosphatidylcholine acyltransferases (LPCATs) proteins from the diatom P. tricornutum, we performed the search of the P. tricornutum genome database using the Basic Local Alignment Search Tool (BLAST) program with the yeast and Arabidopsis LPCAT acyltransferase sequences
The search for the putative signal sequences using PredSL and PSORTII showed that PtLPCAT1 has a predicted N-terminal signal peptide and localization in the endoplasmic reticulum (ER) and plasma membrane (44% for the k-NN prediction)
Summary
To identify the putative LPCAT proteins from the diatom P. tricornutum, we performed the search of the P. tricornutum genome database using the BLAST program with the yeast and Arabidopsis LPCAT acyltransferase sequences. Phylogenetic analysis was performed with amino acid sequences of putative LPCATs from yeast, diatom, mouse, human, and plants. The phylogenetic tree showed that the analyzed LPCATs are related to a broad range of sequences, i.e., P. tricornutum PtLPCAT1 is most closely related to the yeast LPCATs from S. cerevisiae and S. pombe, followed by two human and mouse LPCAT4 proteins, which form clusters with plant LPCATs from A. thaliana and H. annuus. Further increases of microsomal membrane concentration to 0.5, 1, and 2 nmol of microsomal PC/assay caused a progressive reduction in the activity of the tested enzyme to 41%, 20%, and 10% (respectively) of its maximum level (Figure 2a). Phylogramofof the the LPCAT family showing proteins from diatom, yeast, human, mouse, and plants. LPCAT family showing 16 proteins from diatom, yeast, human, mouse, and plants.
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