Phosphorylate ethanolamine

Figure 12.12 Sphingomyelins. Sphingomyelins are esters of a ceramide and phospho-ryl choline. However, similar compounds are ceramide-1-phosphoryl ethanolamines and phosphono forms of sphingolipids. Ceramides W-acyl-sphingosines) are amides of a long chain di- or trihydroxy base containing 12 to 22 carbon atoms, of which sphingosine (4-sphingenine) is the commonest, and a long chain fatty acid whose acyl chain is shown by R1. This may contain up to 26 carbon atoms.

The majority of PC is synthesized in mammalian cells by the CDP-choline or Kennedy pathway in the endoplasmic reticulum (Eigiue 1). In this pathway, choline taken up from the external medium or released in the cytosol by breakdown of choline containing compoimds, is first converted to phosphocholine by the enzyme choline kinase (CK) (Ishidate, 1997). There are two isoforms of CK cloned which both can convert also ethanolamine to phosphoethanolamine, albeit with a lesser affinity (Aoyama et al 2000). Alternatively phosphocholine can be generated by enzymes that preferentially phosphorylate ethanolamine and are therefore designated ethanolamine kinases (EK). As yet also two different EKs are known (EKI 1 and 2 Lykidis etal., 2001). 

ET), which catalyzes the formation of CDP-ethanolamine, and (iii) an ethanolaminephosphotransferase (EPT), which finally synthesizes PE from DAG and CDP-ethanolamine. As discussed rmder PC synthesis, four enzymes have been cloned that can phosphorylate ethanolamine, two of which preferentially use ethanolamine as a substrate, and two which are more specific for choline. Only one isoform of ET has been cloned, which contains two active sites, but seems to be not as strictly regulated compared to its counterpart CT (Bladergroen et al, 1999a). 

This enzyme [EC 2.7.7.14], also referred to as ethanol-amine-phosphate cytidylyltransferase and phosphoryl-ethanolamine transferase, catalyzes the reaction of CTP with ethanolamine phosphate to produce CDP-ethanol-amine and pyrophosphate. 

With the establishment of the phosphoryl enzyme, the question was whether or not the phosphoryl enzyme was the same as the phospho-protein found by incubating inorganic phosphate with alkaline phosphatase at low pH (35, 114-116, 119, 120). Wilson and Dayan (105) pointed out that the phosphoprotein is thermodynamically very stable It is 105 times more stable than O-phosphorylserine (125) and 0-phosphoryl ethanolamine (105, 126). Alkaline phosphatase, as a true catalyst, must catalyze both the hydrolysis and the formation of phosphate esters. Therefore, if a serine residue existed which was capable of forming a thermodynamically stable phosphate ester, alkaline phosphatase as a nonspecific catalyst would catalyze its formation from both inorganic phosphate and phosphoester substrates. 

Structural analysis of LOS from a mutant of Xcc defective in core completion revealed that this mutant had modifications in the lipid A moiety, which had reduced acylation and was further derivatised with phosphoryl ethanolamine residues (Dow et al., 1995 Silipo et al., 2008). These changes in lipid A structure abolished the ability to trigger innate immune responses in Arabidopsis (Silipo et al., 2008). Importantly these findings indicate that Xcc has the capacity to modify the structure of its lipid A to reduce its activity as a MAMP in plants (Silipo et al., 2008). It is not known whether these (or other) modifications to lipid A occur when bacteria are within plants. 

The conversion of choline to choline-P was first demonstrated in yeast extracts by Wittenberg and Komberg (more famous for his contributions to DNA replication) in 1953. The enzyme was purified (K. Ishidate, 1984) from rat kidney and shown also to phosphorylate ethanolamine [5]. This kinase is now referred to as choline kinase p. Three isoforms of choline kinase have been identified (al oi2, and P). Northern analyses indicate that the mRNA encoding choline kinase al is most abundant in testis. Choline kinase o2 is a splice variant of choline kinase al. The choline kinase a and P genes (Chka and Chkb, respectively) have been characterized. The length of Chka is 40 kb whereas Chkb is only... 

The available methods are suitable only for fractionation according to the basic moieties, whereas phosphatides differing in the lipid moieties are not separated from each other. Compounds which contain differing functional groups in the lipid moiety can be indirectly separated from each other using the following procedure [174] the enzyme phospholipase C from bacteria [Clostridium perfringens or Bacillus cereus) splits off phosphoryl ethanolamine and phosphoryl choline from the phosphatides. The lipophilic hydrolysis products can then be separated as acetyl derivatives (I, II, III) on adsorbent layers. 

Glycerophosphate and phosphoryl ethanolamine containing were prepared by Chaikoff et al, (32), and their incorporation into phosphatides of liver and kidney was demonstrated by surviving slices and also in the intact animal. These experiments gave no proof that breakdown of these labeled compounds to inorganic phosphate did not occur before conversion of the radioactive phosphorus to phosphatide. 

Phosphatidylethanolamine synthesis begins with phosphorylation of ethanol-amine to form phosphoethanolamine (Figure 25.19). The next reaction involves transfer of a cytidylyl group from CTP to form CDP-ethanolamine and pyrophosphate. As always, PP, hydrolysis drives this reaction forward. A specific phosphoethanolamine transferase then links phosphoethanolamine to the diacylglycerol backbone. Biosynthesis of phosphatidylcholine is entirely analogous because animals synthesize it directly. All of the choline utilized in this pathway must be acquired from the diet. Yeast, certain bacteria, and animal livers, however, can convert phosphatidylethanolamine to phosphatidylcholine by methylation reactions involving S-adenosylmethionine (see Chapter 26). 

In a study of the attack of the ambident nucleophile ethanolamine on a series of phosphorylating agents (48) and related compounds it was observed that the proportion of O-phosphorylation increased as R and R varied in the series McaN, RO, R and as X varied in the series R2PO, CN, F, this last giving exclusive 0-phosphorylation. These results were... 

Analogues of phosphatidyl ethanolamine, phosphatidyl choline, and phosphatidic acid with pantoic acid skeleton were prepared by phosphorylation with the respective phosphoric mono-, bis-, and tristriazolide [17]... 

Phosphatidylcholine (lecithin) is the most abundant phospholipid in membranes. Phosphatidylethanolamine (cephalin) has an ethanolamine residue instead of choline, and phosphatidylserine has a serine residue. In phosphatidylinositol, phosphatidate is esterified with the sugarlike cyclic polyalcohol myo-inositol. A doubly phosphorylated derivative of this phospholipid, phosphatidylinositol 4,5-bisphosphate, is a special component of membranes, which, by enzymatic cleavage, can give rise to two second messengers, diacylglycerol (DAG) and inositol l,4,5trisphosphate (InsPsi see p.386). 

This enzyme [EC 2.7.1.32] catalyzes the phosphoryl transfer from ATP with choline to produce ADP and O-phosphocholtne. Ethanolamine and its methyl and ethyl derivatives can also serve as substrates. 

This enzyme [EC 2.7.1.82] catalyzes the ATP-dependent phosphorylation of ethanolamine to yield O-phospho-ethanolamine and ADP. 

Synthesis of PE and PC from preexisting choline and ethanolamine These synthetic pathways involve the phosphorylation of choline or ethanolamine by kinases, followed by conversion to the activated form, CDP-choline or CDP-ethanolamine. Finally, choline-phosphate or ethanolamine-phosphate is transferred from the nucleotide (leaving CMP) to a molecule of diacylglycerol (see Figure 17.5). 

Choline and ethanolamine are activated in much the same way as are sugars. For example, choline can be phosphorylated using ATP (Eq. 17-58, step a) and the phosphocholine formed can be further converted (Eq. 17-58, step b) to cytidine diphosphate choline. Phosphocholine is transferred from the latter onto a suitable acceptor to form the final product (Eq. 17-58, step c). Tire polymerization pattern differs from that for polysaccharide synthesis. When the sugar nucleotides react, the entire nucleoside diphosphate is eliminated (Eq. 17-56), but CDP-choline and CDP-ethanolamine react with elimination of CMP (Eq. 

Fig. 21-5, are also used for formation of both phosphatidylcholine and phosphatidylethanolamine. In both cases, the free base, choline, or ethanolamine180a b is phosphorylated with ATP. Choline phosphate formed in this manner is then converted by reaction with CTP to CDP-choline (Eq. 17-58).181 Phosphatidylcholine is formed from this intermediate1813/b while CDP-ethanolamine is used to form phosphatidylethanolamine (Fig. 21-5). These synthetic reactions occur within cell nuclei as well as on surfaces of cytoplasmic membranes.1810...

Scheme I shows the hydrolysis of a phosphate ester in the presence of tris, which can serve as a phosphate acceptor so that O-phosphoryl-tris is a product as well as P(. It has been shown that in the presence of alcohols such as tris and ethanolamine the rate of substrate utilization is increased, that formation of alcohol exceeds that of phosphate, and that the difference is due to the formation of the O-phosphorylamino alcohol (122, 128). The question was Does the reaction with water and with tris emanate from the Michaelis complex or from a phosphoryl enzyme intermediate (E-P) If the reactions with tris and water stem from a phosphoryl enzyme, the ratio of products tris-phosphate and Pi would be independent of the leaving group RO, but if the reactions stem from the reversible complex containing the leaving group, the ratio of products would depend upon the structure of R. It was found that the ratio of free alcohol to phosphate was 2.39 0.02 for nine different substrates, including esters such as p-cresyl phosphate / -naphthyl phosphate, and phosphoenol pyruvate. This experiment established the occurrence of a phosphoryl enzyme intermediate.

The second phase of phospholipid synthesis in eukaryotes. Choline or ethanolamine enters the cell via active transport mechanisms and is immediately phosphorylated by the enzyme, choline (ethanolamine) kinase. The phosphorylated derivatives of choline and ethanolamine... 

The presence in bacterial lipopolysaccharides of phosphoryl-ated ethanolamine residues (1) prompted us to experiment with the incorporation of this group. When compound was treated with an activated phosphorylethanolamine derivative, prepared by the procedure shown in Figure 8, the a-phosphate [a]j) +66.7° (chloroform), was obtained in 46% yield. The reaction is similar to that with dibenzyl tributylstannyl phosphate. Thus, the new phosphorylation method may have wide application in the synthesis of C-l phosphorylated lipid A analogs. 

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