Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast

Abstract

The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O,18O]phosphoethanolamine ([17O,18O]DPPE) were synthesized with slight modification of the previous procedure [Bruzik, K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754] and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-[16O,17O,18O]phosphoric acid ([16O,17O18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O,17O,18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O,18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O,18O]CDP-DPG with a mixture of L-serine, PS synthase (which converted [17O,18O]CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave [17O,18O]DPPE. The configuration and isotopic enrichments of the starting [17O,18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.

Isolation of a Chinese Hamster Ovary (CHO) cDNA Encoding Phosphatidylglycerophosphate (PGP) Synthase, Expression of Which Corrects the Mitochondrial Abnormalities of a PGP Synthase-defective Mutant of CHO-K1 Cells

“…3, and the other has a consensus motif, DXXDGXXARXXXXXXXXGXXLDXXXD (42), that is also found in E. coli PGP synthase, yeast phosphatidylinositol synthase, yeast phosphatidylserine synthase, and yeast CL synthase. This structural difference may reflect a difference in the reaction mechanism between these two groups of enzymes (43). The reaction mechanism of E. coli phosphatidylserine synthase (HKD motif), which shares homology with both yPGS1 and cPGS1, has been proposed to proceed via a ping-pong reaction mechanism involving a phosphatidyl-enzyme intermediate.…”

“…The reaction mechanism of E. coli phosphatidylserine synthase (HKD motif), which shares homology with both yPGS1 and cPGS1, has been proposed to proceed via a ping-pong reaction mechanism involving a phosphatidyl-enzyme intermediate. On the other hand, yeast phosphatidylserine synthase (alternate motif), which shares homologous with the E. coli PGP synthase, appears to proceed by a single displacement sequential Bi-Bi reaction mechanism (43). Based on these sequence homologies, the eukaryotic PGP synthases most likely utilize a ping-pong reaction mechanism, in contrast to the highly homologous prokaryotic PGP synthases (44) that employ a Bi-Bi reaction mechanism.…”

The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily

“…Early evidence showing retention of configuration at the substrate phosphorous in the reactions catalyzed by cabbage PLD and Escherichia coli phosphatidylserine synthase suggested a ''ping pong''-type mechanism with the formation of a covalent phosphoenzyme intermediate (6)(7)(8). The direct demonstration of such a covalent intermediate for S. typhimurium Nuc and Yersinia pestis murine toxin substantiates this view and reveals that the active site nucleophile in the reaction is one of the highly conserved histidines of the HKD motif (9,10).…”

“…Early evidence showing retention of configuration at the substrate phosphorous in the reactions catalyzed by cabbage PLD and Escherichia coli phosphatidylserine synthase suggested a ''ping pong''-type mechanism with the formation of a covalent phosphoenzyme intermediate (6)(7)(8)…”

C‐terminal loop of Streptomyces phospholipase D has multiple functional roles

“…Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062537907. (Stanacev and StuhneSekalec 1970;Bruzik and Tsai 1984;Raetz et al 1987;Gottlin et al 1998). In particular, Streptomyces PLDs show a higher transphosphatidylation activity than those from many other sources (Juneja et al 1988;Hagishita et al 2000).…”