Literature summary for 2.3.1.269 extracted from

Wiseman, B.; Hoegbom, M.
Conformational changes in Apolipoprotein N-acyltransferase (Lnt) (2020), Sci. Rep., 10, 639 .

Search for more literature for 2.3.1.269

Cloned(Commentary)

Cloned (Comment)	Organism
gene lnt, recombinant expression of C-terminally His8-tagged enzyme in Escherichia coli strain C41(DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant C-terminally His8-tagged enzyme, 15 mg/ml protein in 25 mM Tris, pH 8.0, 100 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 0.02% DDM, is mixed with 25% PEG 2000 MME, 100 mM Na cacodylate, pH 6.4, and 40 mM MgCl2 with a drop ratio of 1:1.75 protein:precipitate, 18°C, 2 weeks. For the N-terminally cleaved detagged enzyme, 36% PEG 200, 400 mM ammonium phosphate dibasic, and 100 mM HEPES, pH 7.2, at 10°C for 3 weeks, are used. X-ray diffraction structure determination and analysis at 3.1-3.5 A resolution, modeling	Escherichia coli

Crystallization (Comment)

Organism

purified recombinant C-terminally His8-tagged enzyme, 15 mg/ml protein in 25 mM Tris, pH 8.0, 100 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 0.02% DDM, is mixed with 25% PEG 2000 MME, 100 mM Na cacodylate, pH 6.4, and 40 mM MgCl2 with a drop ratio of 1:1.75 protein:precipitate, 18°C, 2 weeks. For the N-terminally cleaved detagged enzyme, 36% PEG 200, 400 mM ammonium phosphate dibasic, and 100 mM HEPES, pH 7.2, at 10°C for 3 weeks, are used. X-ray diffraction structure determination and analysis at 3.1-3.5 A resolution, modeling

Escherichia coli

Localization

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

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	-	a 1-lyso-phosphoglycerolipid + a [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	?

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P23930	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His8-tagged enzyme from Escherichia coli strain C41(DE3) membrane fraction by nickel affinity chromatography, ultrafiltration, gel filtration, and again ultrafiltration	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	-	?
a phosphoglycerolipid + an [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine	Lnt can use all available phospholipids such as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), and cardiolipin (CL) as acyl donors. The preferred substrate is PE	Escherichia coli	a 1-lyso-phosphoglycerolipid + a [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	?
cardiolipin + [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	Escherichia coli	1-lyso-cardiolipin + [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	?
additional information	the short loop containing W237 might help to locally stabilize the N-terminal tail from the opposite side to allow transfer of the palmitoylate from the C387 to the apolipoprotein to produce the final mature lipoprotein	Escherichia coli	?	-	-
phosphatidylethanolamine + [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	Escherichia coli	1-lyso-phosphatidylethanolamine + [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	?
phosphatidylglycerol + [apolipoprotein]-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	Escherichia coli	1-lyso-phosphatidylglycerol + [lipoprotein]-N-acyl-S-1,2-diacyl-sn-glyceryl-L-cysteine	-	?

Synonyms

Synonyms	Comment	Organism
lnt	-	Escherichia coli

General Information

General Information	Comment	Organism
evolution	apolipoprotein N-acyltransferase (Lnt) belongs to the nitrilase superfamily. Nitrilases are multimeric proteins that contain a common Glu-Lys-Cys catalytic triad that hydrolyse carbon-nitrogen bonds	Escherichia coli
additional information	two crystal forms of Lnt from Escherichia coli are observed. In one form a highly dynamic arm occurs that is able to restrict access to the active site as well as a covalent modification to the active site cysteine consistent with the thioester acyl-intermediate. In the second form, the enzyme crystallizes in an open conformation exposing the active site to the environment. Three unique Lnt molecules that when taken together suggest the movement of essential loops and residues are triggered by substrate binding that could control the interaction between Lnt and the incoming substrate apolipoprotein, mechanism, detailed overview. In the case of Lnt, the nitrilase domain catalyzes the attachment of a fatty acid derived from a phospholipid to the alpha-amino group of the N-terminal cysteine of the apolipoprotein creating the final mature lipoprotein. This attachment occurs via a proposed 2-step ping-pong mechanism where the first step is the acyl transfer of the phospholipid substrate to create a thioester linkage on the active site cysteine. The second step is the transfer of the acyl chain from this cysteine to the N-terminal cysteine of the apolipoprotein. This occurs at the catalytic triad of E267-K335-C387 where E267 acts as a general base to activate the nucleophile of the thiol group of C387 that can then attack the ester linkage between the acyl chain and the glycerol backbone of the phospholipid substrate to form the thioester acyl intermediate. K335 provides part of the oxyanion hole to stabilize this tetrahedral intermediate of the reaction. In the second step, with Lnt now in its thioester-acyl intermediate state, the alpha-amino group at the N-terminus of the incoming apolipoprotein attacks the thioester linkage to transfer the acyl chain to produce the final mature lipoprotein. Similar to the first step, K335 stabilizes the tetrahedral intermediate of the reaction. Docking study and molecular dynamics simulations	Escherichia coli
physiological function	lipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that occurs via a sequential 3-step process controlled by three integral membrane enzymes. The last step of this process, unique to Gram-negative bacteria, is the N-acylation of the terminal cysteine by apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein	Escherichia coli