Identification and Molecular Evolution of Novel Phospholipase D Enzymes in Taxonomically Dispersed Algae using Biocomputational Analyses

BELIGNI, MV; TEN HAVE, ARJEN

Rosario

Congreso; 4to. Congreso Argentina de Bioinformática y Biología Computacional (4CAB2C) y 4ta. Conferencia Internacional de la Sociedad Iberoamericana de Bioinformática (SolBio); 2013

Phospholipase D (PLD) is a key enzyme in phospholipid metabolism that catalyzes the hydrolysis of phosphatidylcholine to form phosphatidic acid (PA), a signaling molecule [1]. Due to the importance of lipid biochemistry in algae and the poor characterization of PA formation in these organisms, we set out to study PLDs in algae and started with a biocomputational meta-analysis. We performed data mining, starting with a high-quality seed dataset of PLDs from reference organisms, by means of iterative HMMer profiling of the complete proteomes from microalgae and related organisms. Figure 1A is a taxonomic tree showing the species used for data mining. Since algae belong to three different kingdoms, representative members of the important taxonomic groups related to algae were included for analysis. The presence of two HxKxxxxD (HKD) domains was used as a preliminary rule for the identification of a protein as a PLD. Sequences containing only one HKD were eliminated in each cycle except for the last one. Sequences with two HKDs (or canonical PLDs, called hereafter PLDs) were separated from sequences with only one HKD domain (PLD homologs). Since complete proteomes for the Rhodophyta and Dinophyta groups are scarce or not available, TBLASTN with genome sequences were performed to augment the information from these two groups. Algae possess less PLDs than higher plants, consistent with their lower signaling complexity. All land plants analyzed have ten PLDs or more, whereas algae typically have one to four, with the exception of the dinoflagellate Alexandrium tamarense, which has at least seven PLDs. Figure 1B shows a phylogenetic tree of PLDs. Plants have mainly C2- and PX-PH-PLDs, based on the regulatory domains they contain. Most algal PLDs belong to the C2 type, while none of the them appear to be PX-PH-PLDs. A few PLDs from chromalveolata algae (diatoms, dinoflagellates, rhizaria) appear to be related to rice signal peptide PLD (SP-PLD) [2]. A few PLDs from A. tamarense are, to our surprise, associated to Phytophthora infestans PLD-like proteins [3]. In addition, a group of PLDs from mamiellophycean green algae (Ostreococcus tauri, Ostreococcus lucimarinus and Micromonas pusilla) are conserved with PLD-like cardiolipin synthases (c-PLDs) from chromalveolata parasites. Hence, it appears that algal PLDs have evolved form a number of ancestors, often but not always explained by their taxonomic classification. A few intriguing ideas arise from this analysis. For example, PLD activity was measured in Chlamydomonas reinhardtii [4], a green alga with no detected PLDs. However, one PLD homolog with a single HKD was identified (data not shown), opening the possibility that some algae might perform the catalytic function of PLDs through homodimeric interaction between PLD homologous polypeptides. 1. Laxalt AM, Munnik T: Phospholipid signalling in plant defence. Curr Opin Plant Biol 2002, 5:332-338. 2. Li G, Lin F, Xue H-W: Genome-wide analysis of the phospholipase D family in Oryza sativa and functional characterization of PLDβ1 in seed germination. Cell Res 2007, 17:881-894. 3. Meijer HJ, Hassen HH, Govers F: Phytophthora infestans has a plethora of phospholipase D enzymes including a subclass that has extracellular activity. PLoS One 2011, 6:e17767. 4. Arisz SA, Valianpour F, van Gennip AH, Munnik T. Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation. Plant J 2003, 34:595-604.