REVIEW Phospholipase D as a catalyst: Application in phospholipid synthesis, molecular structure and protein engineering Jasmina Damnjanovi  c and Yugo Iwasaki * Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received 21 January 2013; accepted 13 March 2013 Available online xxx Phospholipase D (PLD) is a useful enzyme for its transphosphatidylation activity, which enables the enzymatic syn- thesis of various phospholipids (PLs). Many reports exist on PLD-mediated synthesis of natural and tailor-made PLs with functional head groups, from easily available lecithin or phosphatidylcholine. Early studies on PLD-mediated synthesis mainly employed enzymes of plant origin, which were later supplanted by ones from microorganisms, especially acti- nomycetes. Many PLDs are members of the PLD superfamily, having one or two copies of a signature sequence, HxKxxxxD or HKD motif, in the primary structures. PLD superfamily members share a common core structure, and thereby, a common catalytic mechanism. The catalysis proceeds via two-step reaction with the formation of phos- phatidyl-enzyme intermediate. Both of the two catalytic His residues are critical in the reaction course, where one acts as a nucleophile, while the other functions as a general acid/base. PLD is being engineered to improve its activity and stability, alter head group specificity and further identify catalytically important residues. Since the knowledge on PLD enzymology is constantly expanding, this review focuses on recent advances in the field, regarding PLD-catalyzed synthesis of bioactive PLs, deeper understanding of substrate recognition and binding mechanism, altering substrate specificity, and improving thermostability. We introduced some of our recent results in combination with existing facts to further deepen the story on the nature of this useful enzyme. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Phospholipase D; Phospholipid modification; Protein engineering; Thermostability; Substrate specificity] Phospholipase D [EC 3.1.4.4] (PLD) is an enzyme that catalyzes the hydrolysis of the phosphodiester bond of phospholipids (PLs) to generate phosphatidic acid (PA) and an alcohol moiety (Fig. 1A). In addition to the hydrolytic activity, PLD also catalyzes the inter- conversion of polar head groups of PLs, by a process called trans- phosphatidylation (1e3) (Fig. 1B). Transphosphatidylation is especially useful in synthesis of naturally less abundant PLs such as phosphatidylethanolamine (PE), phosphatidylserine (PS), or phos- phatidylglycerol (PG) from highly abundant ones such as phos- phatidylcholine (PC) or lecithin. Synthesized PLs can be used as emulsifiers, components of cosmetics, medical formulations, and for liposome preparations. The main source of lecithin suitable for use in the cost-effective enzymatic bioconversions is soybean oil (4). During the oil refining process, lecithin is produced as a by-product of the crude oil degumming. In this step, addition of water to the crude oil heated at 70e80  C hydrates the PLs, which are then separated from the oil phase by centrifugation, bleached, dried and heat-treated to obtain lecithin. Degumming of other plant seed oils such as corn, cotton- seed, rapeseed, rice or sunflower oil can also yield lecithin. Egg yolk is the major animal source. The main difference between lecithin of plant and animal origin is that the plant-derived lecithin has higher content of unsaturated fatty acids and contains no cholesterol. This review outlines the recent advances in PLD-mediated PL synthesis, molecular structure and protein engineering of microbial PLDs, from the point of their biotechnological applications. Eukaryotic PLDs are key enzymes in the field of PL-mediated cell signaling, for which several recent reviews are available (5e8), and thus, are not covered by this review. PLD-CATALYZED SYNTHESIS OF PHOSPHOLIPIDS Origin of PLDs used in PL modification PLD was first discovered in carrots and cabbage leaves, during the research of Hanahan and Chaikoff in 1940’s (9,10). Later, the PLD activity was observed in a rat brain tissue during the work of Saito and Kanfer (11). However, interest in mammalian PLDs began only after revealing its rapid activation in response to extracellular stimuli (12e14). Up to date, PLDs are found to exist in viruses, prokaryotic, as well as eukaryotic (mammals, yeasts, plants) organisms (5). Since the discovery of plant PLDs with transphosphatidylation activity, the early studies on the PLD-mediated PL modification mainly employed enzymes from cabbage leaves (15). Later on, many researchers isolated PLD-producing microorganisms from soil by screening. Interestingly, most of the isolated strains were * Corresponding author. Tel.: þ81 52 789 4143; fax: þ81 52 789 4145. E-mail addresses: damnjanovic.jasmina@b.mbox.nagoya-u.ac.jp (J. Damnjanovi c), iwasaki@agr.nagoya-u.ac.jp (Y. Iwasaki). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e10, 2013 1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.03.008 Please cite this article in press as: Damnjanovi c, J., and Iwasaki, Y., Phospholipase D as a catalyst: Application in phospholipid synthesis, mo- lecular structure and protein engineering, J. Biosci. Bioeng., (2013), http://dx.doi.org/10.1016/j.jbiosc.2013.03.008