Membrane topology of Escherichia coli diacylglycerol kinase

Smith, Ronald L.; O’Toole, John F.; Maguire, Michael E.; Sanders, Charles R.

doi:10.1128/jb.176.17.5459-5465.1994

Cited by 64 publications

(93 citation statements)

References 37 publications

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“…It was of interest to determine whether the phenotypic effects of the mutation were associated with the lack of or reduction of the enzymatic activity encoded by dgk. DGK from E. coli is an integral membrane protein (27). Therefore, we attempted to detect kinase activity in membrane fractions isolated from S. mutans strains used for the complementation experiments described above.…”

Section: Resultsmentioning

confidence: 99%

“…DGK from Escherichia coli, the only well-characterized member of the family, is an integral membrane protein with a molecular weight of 13,000 containing three predicted transmembrane ␣-helical segments and two amphipathic helices lying along the inner side of the plasma membrane (27). It acts as a homotrimer having three active sites, each consisting of residues belonging to two different subunits (16,34).…”

mentioning

confidence: 99%

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The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

Lis

Kuramitsu

2003

Infect Immun

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We analyzed a previously constructed stress-sensitive Streptococcus mutans mutant Tn-1 strain resulting from disruption by transposon Tn916 of a gene encoding a protein exhibiting amino acid sequence similarity to the Escherichia coli diacylglycerol kinase. It was confirmed that the mutation led to significantly reduced lipid kinase activity, while expression of the intact gene on a plasmid restored both kinase activity and the wild-type phenotype. Further analysis revealed that the product of the dgk gene in S. mutans predominantly recognizes a lipid substrate other than diacylglycerol, most likely undecaprenol, as demonstrated by its efficient phosphorylation and the resistance of the product of the reaction to saponification. The physiological role of the product of the dgk gene as a putative undecaprenol kinase was further supported by a significantly higher sensitivity of the mutant to bacitracin compared with that of the parental strain.A family of bacterial diacylglycerol kinases (DGKs) constitutes a group of highly hydrophobic proteins exhibiting little amino acid sequence similarity to their eukaryotic counterparts. DGK from Escherichia coli, the only well-characterized member of the family, is an integral membrane protein with a molecular weight of 13,000 containing three predicted transmembrane ␣-helical segments and two amphipathic helices lying along the inner side of the plasma membrane (27). It acts as a homotrimer having three active sites, each consisting of residues belonging to two different subunits (16,34). The enzyme catalyzes a direct transfer of phosphate from MgATP to diacylglycerol (2), resulting in the formation of phosphatidic acid (PA). The kinase exhibits broad specificity in vitro for lipid substrates, being able to phosphorylate a variety of diacylglycerols and their analogs, monoacylglycerols, as well as ceramide (5,23,24,36).In bacteria, PA, a major precursor in phospholipid metabolism, is normally synthesized by the sequential acylation of sn-glycero-3-phosphate. PA generation through direct phosphorylation of diacylglycerol by DGK constitutes a minor pathway, which appears to function in the regulation of the diacylglycerol content in the plasma membrane. In E. coli membranes, the diacylglycerol levels are low (approximately 0.5%), yet substantial amounts of neutral lipid are generated during production of membrane-derived oligosaccharide, a periplasmic component involved in osmoregulation. The importance of diacylglycerol recycling via its phosphorylation to PA has been demonstrated in a mutant lacking DGK activity, which was characterized by a marked accumulation of diacylglycerol in the membranes, especially under low osmolarity, a condition stimulating production of membrane-derived oligosaccharide (19,20). In gram-positive bacteria, diacylglycerol is formed as the result of the transfer of sn-glycero-1-phosphate from phosphatidylglycerol to the growing chain of lipoteichoic acid, a cell wall component anchored in the membrane via a glycolipid (10,11,30). The diacylglycero...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

Lis

Kuramitsu

2003

Infect Immun

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“…Phospholipid classes were PtdGro (F), phosphatidylethanolamine (f), lysylphosphatidylglycerol (E), and cardiolipin (Ⅺ). B, pulse-chase labeling of diacylglycerol and phospholipid labeling with [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]acetate. Strain GS435 grown in either the presence (F) or absence (E) of IPTG inducer was pulse-labeled for 10 min at the time point indicated in Fig.…”

Section: Identification Of the B Subtilis Dagk By Genetic Complementmentioning

confidence: 99%

“…The DAG formed is converted to PtdOH for the resynthesis of PtdGro by DgkA (4). DgkA is an inner membrane lipid kinase that exists as a trimer with each monomer containing three membrane-spanning domains (5,6). The physiological substrate for DgkA is DAG, but the enzyme also less efficiently phosphorylates related lipids, like ceramide, that are not found in the bacterium (7,8).…”

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Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis

Jerga

Schujman

et al. 2007

Journal of Biological Chemistry

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Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.Diacylglycerol kinases (DagKs) 5 are key enzymes in phospholipid metabolism that function to reintroduce DAG formed from the breakdown of phospholipids into the biosynthetic pathway. In Escherichia coli, PtdGro is degraded to DAG by the transfer of the sn-glycerol-1-P head group to membrane-derived oligosaccharides

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Rapid and Sequential Dual Oxime Ligation Enables De Novo Formation of Functional Synthetic Membranes from Water‐Soluble Precursors

et al. 2022

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Cell membranes define the boundaries of life and primarily consist of phospholipids. Living organisms assemble phospholipids by enzymatically coupling two hydrophobic tails to a soluble polar head group. Previous studies have taken advantage of micellar assembly to couple single-chain precursors, forming non-canonical phospholipids. However, biomimetic nonenzymatic coupling of two alkyl tails to a polar head-group remains challenging, likely due to the sluggish reaction kinetics of the initial coupling step. Here we demonstrate rapid de novo formation of biomimetic liposomes in water using dual oxime bond formation between two alkyl chains and a phosphocholine head group. Membranes can be generated from non-amphiphilic, water-soluble precursors at physiological conditions using micromolar concentrations of precursors. We demonstrate that functional membrane proteins can be reconstituted into synthetic oxime liposomes from bacterial extracts in the absence of detergent-like molecules.

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Membrane topology of Escherichia coli diacylglycerol kinase

Cited by 64 publications

References 37 publications

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

The Stress-Responsive dgk Gene from Streptococcus mutans Encodes a Putative Undecaprenol Kinase Activity

Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis

Rapid and Sequential Dual Oxime Ligation Enables De Novo Formation of Functional Synthetic Membranes from Water‐Soluble Precursors

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