Transphosphatidylation reaction

Table 2. Comparison of kinetic and selectivity data in the PLD catalyzed transphosphatidylation reaction of PC with primary and secondary alcohols (PLD from S. PMF, ethyl acetate/ace-tate buffer pH 5.5,1 mol L alcohol, 150 mmolL PC, 25 °C, 30 U/g substrate) t, . = time in min of consumption of half of the starting substrate. tf= disappearance of PC (HPLC) [146] ...

Enzymatic conversion of PC to PX via transphosphatidylation reaction of PC has thus become an accepted industrial method for the modification of phospholipids at their polar head. The more frequent and simple application of the biocatalyst consists of the direct use of the culture broth in a biphasic system. The pH optimum and cofactor requirements for some of the most common PLD... 

In almost all of the transphosphatidylation reactions used in the synthesis of novel PLs, PLD from Streptomyces sp. has been used. Several reports showed PLD from cabbage, peanuts, and Streptomyces chromofuscus have low activity for transphosphatidylation [39,40,42,52], although they are able to transphosphatidylate PC to PE and PG. In these few reports, PLD from cabbage catalyzed transphosphatidylation with PC and ethanolamine derivatives [51]. Compared to PLD from Streptomyces sp., PLD from cabbage may possess a more rigid substrate specificity. 

M. Takami and Y. Suzuki Synthesis of novel phosphatidyldihydroxyacetone via transphosphatidylation reaction by phospholipase D. Bioscience, Biotechnology, and Biochemistry 58 (1994) 2136-2139. 

Phospholipase D catalyzes the hydrolysis of phospholipids to produce phosphatidic acid and the corresponding polar head group [215]. In most systems, including neutrophils, phosphotidylcholine is the preferred substrate. Two mammalian isoforms of the enzyme have been cloned, but PLD purified from human neutrophils displays different biochemical characteristics, suggesting it represents a unique isoform [139, 215], In the presence of primary alcohols, PLD catalyzes a transphosphatidylation reaction that produces the corresponding phos-phatidylalcohol. This transphosphatidylation reaction effectively competes with hydrolysis, and thus alcohols such as ethanol and 1-butanol are frequently used experimentally as inhibitors of PLD-catalysed PA production. 

PA is a minor component of the ER membrane that accounts for less than 1% of total ER membrane lipids (Allan, 1996). Formed PA is rapidly consumed by the activity of phosphatidate phosphohydrolase (PAP). In order to measure the formation of PA, the dynamics of PA formation and consumption has to be controlled. This is achieved by exploiting a unique transphosphatidylation reaction that is catalyzed by PLD enzymes. In this reaction, the aliphatic chain of a primary alcohol is transferred to the phosphatidyl moiety of the phosphatidic acid product. In the presence of low concentrations of primary alcohols, PLD enzymes generate phospha-tidylalcohols, which are not recognized by PAP and are not efficiently consumed (Morris et ah, 1997). Therefore the measurement of transphosphatidylation activity of PLD provides a convenient assay that avoids the otherwise highly dynamic nature of the lipid remodeling cascade induced by Sari to support COPII mediated ER export. 

PLD cleaves phosphatidylcholine (PC) to generate choline and phos-phatidic acid (PA) via a transphosphatidylation reaction. However, because PLD is a phosphotransferase, short primary alcohols, such as ethanol, 1-propanol, and 1-butanol, may efficiently substitute for water in the reaction, which then leads to the production of phosphatidylalcohol. 

Such transphosphatidylation reactions were first noticed because phos-phatidylmethanol was formed when plant tissues were extracted with methanol. However, transphosphatidylation does not seem to be a... 

For example, an isozyme of glutathione-S-transferase will also catalyze the fatty acid ethyl-ester synthase reaction, leading to the formation of ethyloleate from oleic acid and ethanol (Bora et al. 1989). Also, phospholipase D catalyzes the transphosphatidylation of phosphatidylcholine with ethanol to form phosphatidylethanol (Kobayashi and Kanfer 1987). The active site requirements and kinetics of the hydrolases or transferases that catalyze these ethylation reactions are not well understood. The elucidation of mechanisms and active site structures for enzyme-catalyzed... 

Phospholipase D (EC 3.1.4.4) is a lipolytic enzyme that hydrolyzes the terminal phosphodiester bond on PLs. Due to its ability to transfer the phosphatidyl moiety of glycerophospholipids to various alcohols (transphosphatidylation), PLD is also used to synthesize PLs with desired head groups that are poorly accessible via the chemical route (Figure 23.4). This ability has been utilized for the synthesis of natural PLs that are rare in nature, such as PG and PS. Novel types of PLs (phosphatidyl-X) have also been synthesized via PLD-mediated transphosphatidylation to add the amphiphilic properties of PLs to the acceptor compounds. These reactions are typically carried out in biphasic systems with water (containing PLD or a hydrophilic alcohol acceptor) and an organic solvent such as chloroform, ether, ethyl acetate, benzene, or toluene. 

PLD-catalyzed transphosphatidylation is a very effective reaction for the preparation of novel PLs with functional moieties. Many compounds were converted to PL derivatives via transphosphatidylation by PLD. Table 23.3 gives the reaction system of novel glycerophospholipids [39-53] the structures of acceptor compounds (alcohols) are presented in Eigure 23.5. 

Ionic calcium is essential for enzyme activity, both for hydrolytic and transphosphatidylation (see below) reactions EDTA inhibits the enzyme in crude extracts. The exact role of Ca has yet to be determined, but there seems little doubt that it is directly involved in the enzyme-phospholipid substrate interaction. 

Whether the aoove reactions play any role in lipid biosynthesis is open to question. The naturally occurring phosphatidylglycerol is the optically active -T-j -glycerol enantiomer. Yang et al. (1967) found that the product of transphosphatidylation was a racemic mixture, although more recently Batrakov et al. (1975) found evidence for stereospecificity in the acyl transfer reaction with phospholipase D. 

Hydrolysis, alcoholysis, esterification, transesterification and transphosphatidylation are some of the reactions used for enzymatic modification of PLs. Enzymatic hydrolysis of PLs is... 

---