Literature summary for 2.7.1.170 extracted from

Bacik, J.P.; Tavassoli, M.; Patel, T.R.; McKenna, S.A.; Vocadlo, D.J.; Khajehpour, M.; Mark, B.L.
Conformational itinerary of Pseudomonas aeruginosa 1,6-anhydro-N-acetylmuramic acid kinase during its catalytic cycle (2014), J. Biol. Chem., 289, 4504-4514.

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Cloned(Commentary)

Cloned (Comment)	Organism
gene anmK, recombinant expression in Escherichia coli strain BL21(DE3) GOLD	Pseudomonas aeruginosa

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme bound to a nonhydrolyzable ATP analogue (AMPPCP) and 1,6-anhydroMurNAc, hanging drop vapor diffusion method, for enzyme AnmK bound to AMPPCP, 6.5 mg/ml protein in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, and 20 mM Tris, pH 8.0, is mixed with an equal volume of mother liquor containing 26% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 9.0, for enzyme AnmK bound to AMPPCP and the anhMurNAc sugar, AnmK s first co-crystallized with AMPPCP by mixing equal volumes of mother liquor containing 24% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 8.2, with the enzyme at 6.5 mg/ml in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, 20 mM Tris, pH 8.0, a single crystal of the AnmK-AMPPCP complex is then transferred to a drop of reservoir buffer supplemented with 8 mM anhMurNAc, 2 mM AMPPCP, and 4 mM MgCl2 and soaked for 1 h, at room temperature, X-ray diffraction structure determination and analysis at 1.67-1.84 A resolution	Pseudomonas aeruginosa

Crystallization (Comment)

Organism

purified recombinant enzyme bound to a nonhydrolyzable ATP analogue (AMPPCP) and 1,6-anhydroMurNAc, hanging drop vapor diffusion method, for enzyme AnmK bound to AMPPCP, 6.5 mg/ml protein in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, and 20 mM Tris, pH 8.0, is mixed with an equal volume of mother liquor containing 26% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 9.0, for enzyme AnmK bound to AMPPCP and the anhMurNAc sugar, AnmK s first co-crystallized with AMPPCP by mixing equal volumes of mother liquor containing 24% PEG 4000, 0.2 M MgCl2, 0.1 M Tris, pH 8.2, with the enzyme at 6.5 mg/ml in 600 mM NaCl, 2 mM AMPPCP, 4 mM MgCl2, 0.5 mM TCEP, 20 mM Tris, pH 8.0, a single crystal of the AnmK-AMPPCP complex is then transferred to a drop of reservoir buffer supplemented with 8 mM anhMurNAc, 2 mM AMPPCP, and 4 mM MgCl2 and soaked for 1 h, at room temperature, X-ray diffraction structure determination and analysis at 1.67-1.84 A resolution

Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, binding structure, overview	Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + 1,6-anhydro-N-acetyl-beta-muramate + H2O	Pseudomonas aeruginosa	-	ADP + N-acetyl-beta-muramate 6-phosphate + H+	-	?
additional information	Pseudomonas aeruginosa	AnmK is an unusual sugar kinase that both cleaves and phosphorylates its 1,6-anhMurNAc substrate	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	Q9I5Q5	gene anmK	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) GOLD by metal affinity chromatography, gel filtration, and ultrafiltration	Pseudomonas aeruginosa

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + 1,6-anhydro-N-acetyl-beta-muramate + H2O = ADP + N-acetylmuramate 6-phosphate	reaction mechanism, structure-function analysis, overview. A one-step catalytic mechanism is proposed that involves attack of water at the anomeric center leading to cleavage of the 1,6-anhydro ring and phosphoryl transfer. The reaction inverts the anomeric center of MurNAc. Transient formation of an intermediate (MurNAc) in a two-step mechanism may also be possible. It is also possible that the gamma-phosphate leaves the ATP as a transient metaphosphate prior to the anhydro-bond cleavage	Pseudomonas aeruginosa	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + 1,6-anhydro-N-acetyl-beta-muramate + H2O	-	Pseudomonas aeruginosa	ADP + N-acetyl-beta-muramate 6-phosphate + H+	-	?
ATP + 1,6-anhydro-N-acetyl-beta-muramate + H2O	sugar substrate binding structure, overview	Pseudomonas aeruginosa	ADP + N-acetyl-beta-muramate 6-phosphate + H+	-	?
additional information	AnmK is an unusual sugar kinase that both cleaves and phosphorylates its 1,6-anhMurNAc substrate	Pseudomonas aeruginosa	?	-	?

Subunits

Subunits	Comment	Organism
More	open and closed ligand-bound enzyme structure, overview	Pseudomonas aeruginosa

Synonyms

Synonyms	Comment	Organism
1,6-anhydro-N-acetylmuramic acid kinase	-	Pseudomonas aeruginosa
AnmK	-	Pseudomonas aeruginosa

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Pseudomonas aeruginosa
additional information	enzyme AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-methylene)triphosphate (AMPPCP)	Pseudomonas aeruginosa

General Information

General Information	Comment	Organism
evolution	althoughAnmKadopts a two-domain fold that is structurally similar to proteins of the hexokinase-hsp70-actin superfamily, 1,6-anhydrosugar kinases are mechanistically unique in that they catalyze both the hydrolysis of the 1,6-anhydro ring and the transfer of the gamma-phosphate group from ATP to O6 of sugar substrates	Pseudomonas aeruginosa
additional information	the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. Ligand binding at the active site of AnmK coordinates its conformational itinerary	Pseudomonas aeruginosa