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Literature summary 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

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.269 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)

EC Number Crystallization (Comment) Organism
2.3.1.269 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

EC Number Protein Variants Comment Organism
2.3.1.269 C23A/C62A site-directed mutagenesis Escherichia coli
2.3.1.269 E389A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
2.3.1.269 F416A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
2.3.1.269 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
2.3.1.269 G342A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
2.3.1.269 additional information generation of a DELTAlnt knockout strain, complementation by expression of the wild-type Lnt enzyme Escherichia coli
2.3.1.269 R352A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
2.3.1.269 V339A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli
2.3.1.269 Y388A site-directed mutagenesis, a Nit domain residue, the mutant cannot complement enzyme-deficient mutant DELTAlnt cells Escherichia coli

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.3.1.269 membrane integral membrane protein Escherichia coli 16020
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.269 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
-
 ?
2.3.1.269 additional information Escherichia coli in vivo activity assay is performed using Para-lntEc Escherichia coli strain PAP8504 ?
-
-

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.269 Escherichia coli P23930
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.269 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.269 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
-
 ?
2.3.1.269 additional information in vivo activity assay is performed using Para-lntEc Escherichia coli strain PAP8504 Escherichia coli ?
-
-

Subunits

EC Number Subunits Comment Organism
2.3.1.269 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

EC Number Synonyms Comment Organism
2.3.1.269 lnt
-
 Escherichia coli

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.1.269 25
-
 assay at, alkylation activity Escherichia coli
2.3.1.269 37
-
 assay at, transacylation activity Escherichia coli

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.1.269 6
-
 assay at, alkylation activity Escherichia coli
2.3.1.269 7.5
-
 assay at, transacylation activity Escherichia coli

General Information

EC Number General Information Comment Organism
2.3.1.269 evolution the enzyme is a member of the nitrilase superfamily which catalyses hydrolysis or condensation of carbon-nitrogen amine and nitrile bonds Escherichia coli
2.3.1.269 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
2.3.1.269 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