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Literature summary for 2.7.1.107 extracted from

  • Li, D.; Stansfeld, P.J.; Sansom, M.S.; Keogh, A.; Vogeley, L.; Howe, N.; Lyons, J.A.; Aragao, D.; Fromme, P.; Fromme, R.; Basu, S.; Grotjohann, I.; Kupitz, C.; Rendek, K.; Weierstall, U.; Zatsepin, N.A.; Cherezov, V.; Liu, W.; Bandaru, S.; English, N.J.; Gati, C.; Barty, A.; Yefanov, O.; Chapman, H.N.; Diederichs, K.; Messerschmidt, M.; Boutet, S.; Williams, G.J.; Seibert, M.M.; Caffrey, M.
    Ternary structure reveals mechanism of a membrane diacylglycerol kinase (2015), Nat. Commun., 6, 10140.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene dgkA, recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain WH1061 Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
purified kinase captured as a ternary complex with bound lipid substrate and an ATP analogue, method optimization, microseeding, the precipitant solution contains 0.2% v/v MPD, 0.1 M NaCl, 0.05 M Na3C6H5O7, pH 5.6, 20°C, X-ray diffraction structure determination and analysis at resolutions of 2.18-3.20 A Escherichia coli

Protein Variants

Protein Variants Comment Organism
A100L site-directed mutagenesis, inactive mutant Escherichia coli
A13K site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme Escherichia coli
A13R site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
A30L site-directed mutagenesis, the mutant shows 93% reduced activity compared to the wild-type enzyme Escherichia coli
D80A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
D80E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
D80N site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
D81A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
D81K site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
D95A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
D95E site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
D95N site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme Escherichia coli
E28A site-directed mutagenesis, the mutation principally affects the binding of the Zn2+ ion, In the absence of the E28 side chain the zinc ions become purely coordinated by E76 and the ATP phosphates, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
E28D site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
E28N site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
E28Q site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
E28R site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
E34A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
E34D site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
E34Q site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
E69A site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
E69D site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
E69Q site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
E76A site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
E76A site-directed mutagenesis, the mutation principally affects the binding of the Zn2+ ion Escherichia coli
E76D site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
E76Q site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
G20A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
G83P site-directed mutagenesis, inactive mutant Escherichia coli
G97P site-directed mutagenesis, inactive mutant Escherichia coli
K94A K94 coordinates both alpha-phosphate and N7 of the adenine ring of ATP, the loss of the basic side-chain releases the adenine of ATP and the binding is lost, almost inactive mutant Escherichia coli
K94M K94 coordinates both alpha-phosphate and N7 of the adenine ring of ATP, the loss of the basic side-chain releases the adenine of ATP and the binding is lost, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
K94R K94 coordinates both alpha-phosphate and N7 of the adenine ring of ATP, the loss of the basic side-chain releases the adenine of ATP and the binding is lost, almost inactive mutant Escherichia coli
N72A site-directed mutagenesis, inactive mutant, Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 Escherichia coli
N72D site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
N72Q site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
R32A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
R32K site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
R9A site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
R9E site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
R9H site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
R9K site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Escherichia coli
S17A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
S73A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Escherichia coli
S90P site-directed mutagenesis, inactive mutant Escherichia coli
S98A site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme Escherichia coli
Y86A site-directed mutagenesis, the mutant shows unaltered activity compared to the wild-type enzyme Escherichia coli
Y86F site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
plasma membrane the enzyme is an integral membrane protein, a trimer situated with half its bulk in the membrane and half in the cytosol Escherichia coli 5886
-

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Escherichia coli
Zn2+ required Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
ATP + 1,2-diacyl-sn-glycerol Escherichia coli
-
ADP + 1,2-diacyl-sn-glycerol 3-phosphate
-
?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0ABN1 gene dgkA
-

Purification (Commentary)

Purification (Comment) Organism
gene dgkA, recombinant N-terminally His-tagged wild-type and mutant enzymes from Escherichia coli strain WH1061 by nickel affinity chromatography and gel filtration. The size-exclusion chromatography step has no effect on specific activity Escherichia coli

Reaction

Reaction Comment Organism Reaction ID
ATP + 1,2-diacyl-sn-glycerol = ADP + 1,2-diacyl-sn-glycerol 3-phosphate analysis of the catalytic mechanism using crystal structure anaysis, structure-function analysis, mutagenesis studies, molecular dynamics simulations, and density functional theory modelling, overview Escherichia coli

Source Tissue

Source Tissue Comment Organism Textmining

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + 1,2-diacyl-sn-glycerol
-
Escherichia coli ADP + 1,2-diacyl-sn-glycerol 3-phosphate
-
?

Subunits

Subunits Comment Organism
trimer the enzyme crafts three catalytic and substrate-binding sites centred about the membrane/cytosol interface Escherichia coli

Synonyms

Synonyms Comment Organism
DGK
-
Escherichia coli
DGKA
-
Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.9
-
assay at Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
Escherichia coli

General Information

General Information Comment Organism
additional information Asn72 plays a key role in catalysis. Its side-chain amide bridges Glu69 and Glu76 both of which are essential, the gamma-phosphate of ATP is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane, catalytic mechanism, overview. The putative catalytic site resides on the protein at the membrane/cytosol interface where the reactive moieties of the two substrates, with disparate polarities, come together for reaction. The ternary complex site, asBC, contains zinc-ACP and two lipid substrates. The gaamma-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane Escherichia coli