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 (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 | 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 Maximum [Da] | Comment | Organism |
---|---|---|---|
38800 | - |
SDS-PAGE, Hph5 protein (341 residues and 6His) | Escherichia coli |
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 | 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 (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 | Comment | Organism | Textmining |
---|---|---|---|
cell culture | strain B834 | Escherichia coli | - |
cell culture | strain BL21 | Escherichia coli | - |
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 | Comment | Organism |
---|---|---|
HPH | - |
Escherichia coli |
Hph5 | - |
Escherichia coli |
hygromycin B phosphotransferase | - |
Escherichia coli |
hygromycin-B kinase | - |
Escherichia coli |
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 | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Escherichia coli |