Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Abstract

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.

Novel processive and nonprocessive glycosyltransferases from Staphylococcus aureus and Arabidopsis thaliana synthesize glycoglycerolipids, glycophospholipids, glycosphingolipids and glycosylsterols

“…Tsai and coworkers who synthesized chiral nucleotides, in which a P‐O bond was substituted by P‐S or P‐O‐isotopes ( 17 O or 18 O). These stereochemically defined phospholipids were found to be useful in studies of the stereochemical mechanisms of reactions catalyzed by phospholipase A 2 [34,35] and the formation of inositol 1‐phosphate catalyzed by phospholipase C [36]. In the present work, we describe, for the first time, preparation of chiral phospholipids PL1 Ac,Me and PL2 Ac,Me by derivatization of two natural prochiral phospholipids PL1 and PL2, which were synthesized by the novel glycosyltransferase from S. aureus expressed in E. coli BL21 (DE3).…”

Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

“…Early studies using strategically radiolabeled substrates and intermediates suggested that PLD enzymes use a two-step "ping pong" reaction mechanism in which a covalent phosphoenzyme intermediate is created before release of the product (Stanacev and Stuhne-Sekalec, 1970;Jiang et al, 1984). This reaction mechanism was supported by structural studies using Yersinia pestis murine toxin (Rudolph et al, 1999) and Nuc endonuclease (Gottlin et al, 1998), both members of the HKDcontaining PLD superfamily.…”

The Reaction Mechanism of Phospholipase D from Streptomyces sp. Strain PMF. Snapshots along the Reaction Pathway Reveal a Pentacoordinate Reaction Intermediate and an Unexpected Final Product

“…Other studies indicated that PLD from cabbage retained the configuration of the phosphorus atom in both the hydrolysis and in the transphosphatidylation reaction with different radioactively labelled substrates, all having a chiral phosphorus atom. 5,6,7 In a recent paper, a comparison between PLD activity from two Streptomyces species is presented. 8 Interestingly, one of these PLD enzymes possesses neither of the above-mentioned HxK(x) 4 D (HKD) sequence repeats, resulting in an apparently altered reaction mechanism, which does not proceed through a covalent enzyme -substrate intermediate.…”