Literature summary for 2.7.1.232 extracted from

Bacik, J.P.; Klesmith, J.R.; Whitehead, T.A.; Jarboe, L.R.; Unkefer, C.J.; Mark, B.L.; Michalczyk, R.
Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion (2015), J. Biol. Chem., 290, 26638-26648.

Search for more literature for 2.7.1.232

Cloned(Commentary)

Cloned (Comment)	Organism
functional recombinant expression in Escherichia coli strain BL21(DE3) GOLD, introduced into ethanologenic Escherichia coli by chromosome integration	Lipomyces starkeyi

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant enzyme, hanging drop vapor diffusion method, dimeric enzyme: mixing of equal volumes of 7 mg/ml protein in 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5mM TCEP, and 20mM Tris, pH 7.5, with reservoir solution containing 18% PEG 4000, 100 mM sodium acetate, 100 mM Tris, pH 7.2, for the free enzyme and with reservoir solution containing 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5 mM TCEP, 1 mM aluminum nitrate, 10 mM sodium fluoride, 20 mM Tris, pH 7.5, for the complexed enzyme. Monomeric enzyme: 9 mg/ml protein in 50 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, and 200 mM levoglucosan is mixed with reservoir buffer containing 1.8 M ammonium sulfate, HEPES, pH 7.0, and 100 mM sodium acetate for th free enzyme, and 25 mg/ml protein in 100 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, 2 mM ADP, 4 mM MgCl2, 1 mM aluminum nitrate, and 10 mM sodium fluoride, with reservoir buffer containing 1.3 M sodium malonate, 93 mM Bis-Tris propane, pH 7.0, for the complexed enzyme, soaking of resultant crystals in the drop with 200 mM levoglucosan. X-ray diffraction structure determination and analysis at 1.5-2.0 A resolution	Lipomyces starkeyi

Crystallization (Comment)

Organism

purified recombinant enzyme, hanging drop vapor diffusion method, dimeric enzyme: mixing of equal volumes of 7 mg/ml protein in 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5mM TCEP, and 20mM Tris, pH 7.5, with reservoir solution containing 18% PEG 4000, 100 mM sodium acetate, 100 mM Tris, pH 7.2, for the free enzyme and with reservoir solution containing 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5 mM TCEP, 1 mM aluminum nitrate, 10 mM sodium fluoride, 20 mM Tris, pH 7.5, for the complexed enzyme. Monomeric enzyme: 9 mg/ml protein in 50 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, and 200 mM levoglucosan is mixed with reservoir buffer containing 1.8 M ammonium sulfate, HEPES, pH 7.0, and 100 mM sodium acetate for th free enzyme, and 25 mg/ml protein in 100 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, 2 mM ADP, 4 mM MgCl2, 1 mM aluminum nitrate, and 10 mM sodium fluoride, with reservoir buffer containing 1.3 M sodium malonate, 93 mM Bis-Tris propane, pH 7.0, for the complexed enzyme, soaking of resultant crystals in the drop with 200 mM levoglucosan. X-ray diffraction structure determination and analysis at 1.5-2.0 A resolution

Lipomyces starkeyi

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required, the enzyme binds two magnesium ions in the active site, four manganese atoms in the dimeric structure, that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. The magnesium ions are observed in ideal octahedral coordination with Glu362 and Asp26, several water molecules, and the ADP, overview. The first of the bound metals, designated M1, forms an electrostatic interaction with the beta-phosphate, and its positioning suggests that it plays a direct role in phosphoryl transfer. The second of these metals, designated M2, likely plays a role in coordinating the position of the alpha- and beta-phosphates because it binds to both of these phosphates, whereas a role in modulation of electrostatic charges is also plausible	Lipomyces starkeyi

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
ATP + levoglucosan + H2O	Lipomyces starkeyi	-	ADP + D-glucopyranose 6-phosphate	-	?
ATP + levoglucosan + H2O	Lipomyces starkeyi YZ-215	-	ADP + D-glucopyranose 6-phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Lipomyces starkeyi	B3VI55	-	-
Lipomyces starkeyi YZ-215	B3VI55	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose	mechanism of 1,6-anhydro bond cleavage, the enzyme binds levoglucosan in two distinct orientations, in addition to cleavage of the 1,6-anhydro ring, the pyranose ring of the levoglucosan is opened when the anomeric carbon of levoglucosan migrates from its position in a 1C4 to a 4C1 conformation, increased conformational strain during this reaction results in the linear form of G6P as the initial product, which then equilibrates to a mixture of both anomeric forms, structure-function analysis, detailed overview	Lipomyces starkeyi	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
ATP + levoglucosan + H2O	-	Lipomyces starkeyi	ADP + D-glucopyranose 6-phosphate	-	?
ATP + levoglucosan + H2O	in addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom	Lipomyces starkeyi	ADP + D-glucopyranose 6-phosphate	-	?
ATP + levoglucosan + H2O	-	Lipomyces starkeyi YZ-215	ADP + D-glucopyranose 6-phosphate	-	?
ATP + levoglucosan + H2O	in addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom	Lipomyces starkeyi YZ-215	ADP + D-glucopyranose 6-phosphate	-	?
additional information	although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK, EC 2.7.1.170), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. A second alternate binding orientation for levoglucosan is found, and in these structures, ADP binds with lower affinity, explaining the high Km of enzyme LGK for levoglucosan. LGK binds the reaction product ADP through multiple hydrogen bonds, including bonds between the adenyl moiety and Asp237, between the nucleotide ribose and Asp221, and between the ADP phosphates and protein residues Ser24, Gly189, and Gly328, mechanism of 1,6-anhydro bond cleavage, overview	Lipomyces starkeyi	?	-	?
additional information	although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK, EC 2.7.1.170), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. A second alternate binding orientation for levoglucosan is found, and in these structures, ADP binds with lower affinity, explaining the high Km of enzyme LGK for levoglucosan. LGK binds the reaction product ADP through multiple hydrogen bonds, including bonds between the adenyl moiety and Asp237, between the nucleotide ribose and Asp221, and between the ADP phosphates and protein residues Ser24, Gly189, and Gly328, mechanism of 1,6-anhydro bond cleavage, overview	Lipomyces starkeyi YZ-215	?	-	?

Synonyms

Synonyms	Comment	Organism
levoglucosan kinase	-	Lipomyces starkeyi
LGK	-	Lipomyces starkeyi

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Lipomyces starkeyi

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.6	9	assay at	Lipomyces starkeyi

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Lipomyces starkeyi