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Literature summary for 2.3.1.269 extracted from

  • Lu, G.; Xu, Y.; Zhang, K.; Xiong, Y.; Li, H.; Cui, L.; Wang, X.; Lou, J.; Zhai, Y.; Sun, F.; Zhang, X.C.
    Crystal structure of E. coli apolipoprotein N-acyl transferase (2017), Nat. Commun., 8, 15948 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.3.1.269

Cloned(Commentary)

Cloned (Comment) Organism
gene lnt, recombinant expression of His-tagged wild-type enzyme in Escherichia coli strain C41(DE3), the His-tagged selenomethionine (Se-Met) labeled Lnt is expressed in Escherichia coli methionine-auxotrophy strain B834 (DE3), recombinant expression of enzyme point mutants in Escherichia coli DELTAlnt knockout strain for complementation studies Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
purified recombinant wild-type and selenomethionine-labeled enzymes, hanging drop vapor diffusion method, 12 mg/ml protein from 50 mM Tris-HCl, pH 7.5, and 26% v/v PEG 550 MME supplemented with two detergents, n-heptyl-beta-D-thioglucopyranoside and CHAPSO, 16°C, X-ray diffraction structure determination and analysis at 2.59 and 3.60 A resolution, respectively Escherichia coli

Protein Variants

Protein Variants Comment Organism
C23A/C62A site-directed mutagenesis Escherichia coli
E389A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
F416A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
G145A site-directed mutagenesis, G145 is located in a highly conserved region C-terminal to TM5, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
G342A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
additional information generation of a DELTAlnt knockout strain, complementation by expression of the wild-type Lnt enzyme Escherichia coli
R352A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
V339A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
Y388A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane integral membrane protein Escherichia coli 16020
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Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
a phosphoglycerolipid + an [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine Escherichia coli
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 a 1-lyso-phosphoglycerolipid + a [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine
-
 ?
additional information Escherichia coli in vivo activity assay is performed using Para-lntEc Escherichia coli strain PAP8504 ?
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-

Organism

Organism UniProt Comment Textmining
Escherichia coli P23930
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-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type enzyme from Escherichia coli strain C41(DE3) and His-tagged Se-Met-labeled Lnt from Escherichia coli strain B834 (DE3), both by nickel affinity chromatography, desalting gel filtration, ion exchange chromatography, and ultrafiltration and gel filtration Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
a phosphoglycerolipid + an [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine
-
 Escherichia coli a 1-lyso-phosphoglycerolipid + a [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine
-
 ?
additional information in vivo activity assay is performed using Para-lntEc Escherichia coli strain PAP8504 Escherichia coli ?
-
-

Subunits

Subunits Comment Organism
More the enzyme contains an exo-membrane nitrilase domain fused to a transmembrane (TM) domain. The TM domain of Lnt contains eight TM helices which form a membrane-embedded cavity with a lateral opening and a periplasmic exit. The nitrilase domain is located on the periplasmic side of the membrane, with its catalytic cavity connected to the periplasmic exit of the TM domain. An amphipathic lid loop from the nitrilase domain interacts with the periplasmic lipid leaflet, forming an interfacial entrance from the lipid bilayer to the catalytic centre for both the lipid donor and acceptor substrates. Crystal structure analysis, overview Escherichia coli

Synonyms

Synonyms Comment Organism
lnt
-
 Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at, alkylation activity Escherichia coli
37
-
 assay at, transacylation activity Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
 assay at, alkylation activity Escherichia coli
7.5
-
 assay at, transacylation activity Escherichia coli

General Information

General Information Comment Organism
evolution the enzyme is a member of the nitrilase superfamily which catalyses hydrolysis or condensation of carbon-nitrogen amine and nitrile bonds Escherichia coli
metabolism in Gram-negative bacteria, lipid modification of proteins is catalysed in a three-step pathway. Apolipoprotein N-acyl transferase (Lnt) catalyses the third step in this pathway, whereby it transfers an acyl chain from a phospholipid to the amine group of the N-terminal cysteine residue of the apolipoprotein Escherichia coli
additional information catalytic mechanism two-step reaction catalysed by Lnt, catalytic residue Cys387, the transacylation reaction uses a ping-pong mechanism, structure of the active site with catalytic triad and Glu343, overview. Lnt activity depends on its affinity to the lipid substrate. The enzyme contains an exo-membrane nitrilase domain fused to a transmembrane (TM) domain. The TM domain of Lnt contains eight TM helices which form a membrane-embedded cavity with a lateral opening and a periplasmic exit. The nitrilase domain is located on the periplasmic side of the membrane, with its catalytic cavity connected to the periplasmic exit of the TM domain. An amphipathic lid loop from the nitrilase domain interacts with the periplasmic lipid leaflet, forming an interfacial entrance from the lipid bilayer to the catalytic centre for both the lipid donor and acceptor substrates. Essential Lnt residues are located within the central cavity. Molecular dynamics simulations Escherichia coli