Literature summary for 2.7.1.163 extracted from

LIno, D.; Takakura, Y.; Kuroiwa, M.; Kawakami, R.; Sasaki, Y.; Hoshino, T.; Ohsawa, K.; Nakamura, A.; Yajima, S.
Crystallization and preliminary crystallographic analysis of hygromycin B phosphotransferase from Escherichia coli (2007), Acta Crystallogr. Sect. F, 63, 685-688.

Search for more literature for 2.7.1.163

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Escherichia coli
Expression of hph5 gene in Escherichia coli (strain BL21) and expression of selenomethionine-substituted enzyme in the methionine-auxotroph Escherichia coli strain B834 (DE3)	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging-drop vapour-diffusion method method. The crystals provide diffraction data to a resolution of 2.1 A and belong to space group P3(2)21, with unit-cell parameters a = b = 71.0 A, c = 125.0 A. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP shows the same crystal form as that of the apoprotein	Escherichia coli
using a thermostable mutant and the hanging-drop vapour-diffusion method at 20°C. Thermostable proteins crystallize with less difficulty than wild-type proteins	Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
A118V	mutant enzyme Hph5	Escherichia coli
D20G	mutant enzyme Hph5	Escherichia coli
additional information	mutant enzyme containing all mutations shows an increased thermostability of approximately 16°C K in vivo compared with the wild-type protein	Escherichia coli
Q226L	mutant enzyme Hph5	Escherichia coli
S225P	mutant enzyme Hph5	Escherichia coli
T246A	mutant enzyme Hph5	Escherichia coli

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
38800	-	SDS-PAGE, Hph5 protein (341 residues and 6His)	Escherichia coli

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
hygromycin B + ATP	Escherichia coli	resulting in a loss of cell-kill antibiotic activity of hygromycin B	7''-O-phosphohygromycin B + ADP	-	r

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	-	-	-
Escherichia coli	-	strain BL21 (mutant enzyme Hph5 contains 5 amino-acid substitutions with increased thermostability of approximately -257°C in vivo compared with the wild-type protein), and methionine-auxotroph strain B834 (DE3)	-

Purification (Commentary)

Purification (Comment)	Organism
Harvested cells are suspended in buffer with DNase I and phenylmethylsulfonyl fluoride. The cells are lysed by sonication. Cell debris is separated by centrifugation and the soluble fraction is applied onto an Ni2+-affinity column equilibrated with buffer. The protein is eluted with a linear gradient of imidazole using a fast protein liquid-chromatography system. The eluted protein is dialyzed and applied onto a column of Q-Sepharose FF. The Hph5 protein is eluted with a linear gradient of 0-500 mM NaCl. The purified Hph5 is concentrated to 20-30 mg/ml for crystallization and stored at 20°C. Protein purification is monitored by SDS-PAGE. The selenomethionine-substituted Hph5 is purified using an Ni2+-affinity column and a Q-Sepharose FF column; this process is identical to that uses to purify the native Hph5 and is performed using buffers containing 20 mM mercaptoethanol.	Escherichia coli
recombinant enzyme	Escherichia coli

Purification (Comment)

Organism

Harvested cells are suspended in buffer with DNase I and phenylmethylsulfonyl fluoride. The cells are lysed by sonication. Cell debris is separated by centrifugation and the soluble fraction is applied onto an Ni2+-affinity column equilibrated with buffer. The protein is eluted with a linear gradient of imidazole using a fast protein liquid-chromatography system. The eluted protein is dialyzed and applied onto a column of Q-Sepharose FF. The Hph5 protein is eluted with a linear gradient of 0-500 mM NaCl. The purified Hph5 is concentrated to 20-30 mg/ml for crystallization and stored at 20°C. Protein purification is monitored by SDS-PAGE. The selenomethionine-substituted Hph5 is purified using an Ni2+-affinity column and a Q-Sepharose FF column; this process is identical to that uses to purify the native Hph5 and is performed using buffers containing 20 mM mercaptoethanol.

Escherichia coli

recombinant enzyme

Escherichia coli

Source Tissue

Source Tissue	Comment	Organism	Textmining
cell culture	strain B834	Escherichia coli	-
cell culture	strain BL21	Escherichia coli	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
ATP + hygromycin B	-	Escherichia coli	ADP + 7''-O-phosphohygromycin B	-	?
hygromycin B + ATP	resulting in a loss of cell-kill antibiotic activity of hygromycin B	Escherichia coli	7''-O-phosphohygromycin B + ADP	-	r

Synonyms

Synonyms	Comment	Organism
HPH	-	Escherichia coli
Hph5	-	Escherichia coli
hygromycin B phosphotransferase	-	Escherichia coli
hygromycin-B kinase	-	Escherichia coli

pH Stability

pH Stability	pH Stability Maximum	Comment	Organism
5.1	5.4	Hph5 crystals growing to dimensions of 0.2x0.2x0.2 mm using the conditions 0.1 M sodium acetate, 0.1 M NaCl, 12-20% 2-methylpentane-2,4-diol	Escherichia coli

Cofactor

Cofactor	Comment	Organism	Structure
ATP	-	Escherichia coli