Mechanism of the Enzymatic Synthesis of Cardiolipin in Escherichia coli

Abstract

In previous studies, the enzymatic conversion of phosphatidylglycerol to cardiolipin (diphosphatidylglycerol) in cell-free preparations from E. coli was shown to be stimulated by the addition of CDP-dipalmitin, suggesting the participation of the cytidine coenzyme as phosphatidyl donor. The present communication, however, presents three lines of evidence supporting the following mechanism for the synthesis of cardiolipin in E.coli.2 Phosphatidylglycerol --* cardiolipin + glycerol When CDP-dipalmitin labeled with 32p in the phosphatidyl moiety was incubated with phosphatidyl[2-'HJglycerol, the cardiolipin produced in the enzymatic reaction was labeled with tritium, but not with 32p. Thus, CDP-diglyceride stimulates the reaction but does not participate as phosphatidyl donor. When ['11Pphos-phatidyl[2-3FlHglycerol was used as substrate, the-ratio of tritium to 8p in the cardiolipin product was only half of that in the starting phosphatidylglycerol, consistent with the elimination of 1 mol of glycerol during conversion to cardiolipin. Finally, free glycerol produced during the reaction has been unambiguously identified by phosphorylation with ATP in a reaction catalyzed by glycerol kinase (EC 2.7.1.30), followed by chromatographic isolation of labeled sn"3-glycero-3-phosphate.In a previous communication from this laboratory, Stanacev et al.(1) described a cell-free enzyme preparation from Escherichia coli that catalyzed the formation of cardiolipin (diphosphatidylglycerol) from phosphatidyl [2-8H ]glycerol.The addition of unlabeled CDP-dipalmitin stimulated the conversion of phosphatidylglycerol to cardiolipin 2-to 3-fold. These workers suggested that the synthesis of cardiolipin in this system proceeded according to the following equation: MATERIALS AND METHODSCDP-dipalmitin labeled with a2p in the phosphatidyl moiety was prepared from 1,2-dipalmityl-sn-glycero-3-'2P by an adaptation of the method of Agranoff and Suomi (7). It was purified by chromatography on DEAE-cellulose in chloroform-methanol-water (1), and was shown to be completely reactive enzymatically in an assay with CDP-diglyceride: L-serine phosphatidyltransferase. The labeled phosphatidic acid used in this synthesis was prepared by the enzymatic phosphorylation of 1,2-dipalmityl-atglycerol with [-ys2P]-ATP, by the use of a partially purified, preparation of diglyceride kinase from E. coli kindly provided by E. Gayle Reed. Phosphatidyl[2-'H]glycerol was prepared essentially by the method of Chang and Kennedy (8).[82P]Phosphatidyl-[2-'H]glycerol was similarly prepared with the 82P-labeled CDP-dipalmitin described above.

Absence of Cardiolipin in the crd1 Null Mutant Results in Decreased Mitochondrial Membrane Potential and Reduced Mitochondrial Function

“…This sample was then also incubated with oxygen, and oxidative phosphorylation was started with the substrates. After 2 min, the reaction was quenched by HClO 4 . From the main suspension, aliquots of 0.25 ml were withdrawn and injected into (38).…”

“…Eukaryotes and bacteria utilize different reactions to convert PG to CL. In prokaryotes, CL synthase catalyzes a phosphatidyl transfer between two PG molecules (4). This is a near-equilibrium (transesterification) reaction that is mainly controlled by substrate availability.…”

Cardiolipin is not essential for the growth of Saccharomyces cerevisiae on fermentable or non‐fermentable carbon sources

“…Eukaryotes and bacteria use different reactions to convert PG to CL. In prokaryotes, CL synthase catalyses a phosphatidyl transfer between two PG molecules (Hirschberg and Kennedy, 1972), while in eukaryotes, CL synthase catalyses a phosphatidyl transfer from CDP-DG to PG (Hostetler et al, 1972;Tamai and Greenberg, 1990;Schlame et al, 1993).…”

Specific roles of phosphatidylglycerols in hosts and microbes

“…Three different genes are identified as cardiolipin synthases, named clsA/B/C. In E.coli, the ClsA protein aggregates two PG molecules for cardiolipin formation (24). Some evidences indicate that the other cardiolipin synthases ClsB/ClsC can use PG but also other phospholipid substrates (25).…”