Information on EC 2.7.1.163 - hygromycin B 4-O-kinase

Word Map on EC 2.7.1.163
Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
2.7.1.163
-
RECOMMENDED NAME
GeneOntology No.
hygromycin B 4-O-kinase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + hygromycin B = ADP + 4-O-phosphohygromycin B
show the reaction diagram
-
-
-
-
ATP + hygromycin B = ADP + 7''-O-phosphohygromycin B
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phopho group transfer
-
-
phospho-group transfer
-
-
Phosphorylation
-
-
SYSTEMATIC NAME
IUBMB Comments
ATP:hygromycin-B 4-O-phosphotransferase
Phosphorylates the antibiotic hygromycin B. Whereas the enzyme from Streptomyces hygroscopicus (EC 2.7.1.119; hygromycin-B 7''-O-kinase) catalyses the formation of 7''-O-phosphohygromycin B, this enzyme, found in Escherichia coli carrying a plasmid conferring resistance to hygromycin-B, forms 4-O-phosphohygromycin B.
CAS REGISTRY NUMBER
COMMENTARY hide
88361-67-5
cf. EC 2.7.1.119
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
wild strain T23
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ATP + destomycin A
ADP + 4-O-phosphodestomycin A
show the reaction diagram
-
1-N-methyl isomer of hygromycin B
-
-
?
ATP + destomycin B
ADP + 4-O-phosphodestomycin B
show the reaction diagram
-
1-N-methyl-4',4"-epi hygromycin B
-
-
?
ATP + hygromycin B
ADP + 4-O-phosphohygromycin B
show the reaction diagram
-
phosphorylated product procures hygromcin B resistance
-
-
ir
ATP + hygromycin B
ADP + 7''-O-phosphohygromycin
show the reaction diagram
ATP + hygromycin B2
ADP + 4-O-phosphohygromycin B2
show the reaction diagram
-
pseudodisaccharide of D-talose and hyosamine
-
-
?
hygromycin B + ATP
4-O-phosphohygromycin + ADP
show the reaction diagram
hygromycin B + ATP
7''-O-phosphohygromycin + ADP
show the reaction diagram
-
resulting in a loss of cell-kill antibiotic activity of hygromycin B
-
-
r
additional information
?
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
ATP + hygromycin B
ADP + 4-O-phosphohygromycin B
show the reaction diagram
-
phosphorylated product procures hygromcin B resistance
-
-
ir
ATP + hygromycin B
ADP + 7''-O-phosphohygromycin
show the reaction diagram
-
-
-
-
?
hygromycin B + ATP
4-O-phosphohygromycin + ADP
show the reaction diagram
hygromycin B + ATP
7''-O-phosphohygromycin + ADP
show the reaction diagram
-
resulting in a loss of cell-kill antibiotic activity of hygromycin B
-
-
r
additional information
?
-
-
no phosphorylation of neomycin, kanamycin A and B, streptomycin, dideoxykanamycin B, tobramycin, gentamicin, G418, sisomicin, netilmicin, amikacin, apramycin, ribostamycin, butirosin, lividomycin, and paromomycin by this enzyme
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KCl
-
at 20 mM
NH4+
-
included in assay medium
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2 - 3
ATP
-
HPH and HPH5
0.9 - 2.9
hygromycin B
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5.7 - 6.4
ATP
5 - 9.1
hygromycin B
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
-
purified refolded recombinant enzyme expressed in Escherichia coli
17
-
HPH, ph 7.5 at 30C
21
-
HPH5, ph 7.5 at 30C
35.2
-
HPH5, ph 7.5 at 55C
36.3
-
HPH, ph 7.5 at 50C
additional information
-
107471 counts/minute/25 microl sample extract, hygromycin as substrate; 14328 counts/minute/25 microl sample extract, A23444 as substrate; 15508 counts/minute/25 microl sample extract, SS-56C as substrate; 24483 counts/minute/25 microl sample extract, destomycin A as substrate; 35387 counts/minute/25 microl sample extract, strain BE1065 containing plasmid pKC222; 50051 counts/minute/25 microl sample extract, strain BE1098 containing plasmid pKC241; 5986 counts/minute/25 microl sample extract, hygromycin b2 as substrate; 8428 counts/minute/25 microl sample extract, destomycin B as substrate; hygromycin B is an aminocyclitol antibiotic with broad-spectrum activity against both procaryotic and eucaryotic cells; No activity is found in the plasmid-free DH1 strain.
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
-
assay at
7.8
-
assay at
8
-
assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
-
assay at
50
-
optimum temperature for HPH enzyme activity
55
-
optimum temperature HPH5 enzyme activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
-
x * 38000, recombinant enzyme from transgenic rice plants, SDS-PAGE
38800
-
SDS-PAGE, Hph5 protein (341 residues and 6His)
39030
-
for HPH5, calculated by amino acid sequence and determined by SDS-PAGE
39100
-
for HPH, calculated by amino acid sequence and determined by SDS-PAGE
42000
-
non-dentauring PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
-
x * 38000, recombinant enzyme from transgenic rice plants, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
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; using a thermostable mutant and the hanging-drop vapour-diffusion method at 20C. Thermostable proteins crystallize with less difficulty than wild-type proteins
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
671134
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36
-
HPH, thermal stability of enzyme activity
53
-
HPH5, thermal stability of enzyme activity
55
-
inactivation of wild-type enzyme and mutants S52T and W238C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
protein denaturation of HPH at 37.2C and denaturation of HPH5 at 58.8C
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
from cell culture by centrifugation and sonication, supernatant is loaded onto a column of chelating sepharose fast flow and then proteins are subjected to gel filtration
-
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 20C. 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.; recombinant enzyme
-
recombinant His-tagged enzyme denatured by 8 M urea, by nickel affinity chromatography to over 95% purity showing high immunoactivity
-
soluble renatured recombinant enzyme by anion exchange chromatography to over 95% purity
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Agrobacterium tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase gene under the control of a 35S cauliflower mosaic virus promoter, hygromycin phosphotransferase gene, and neomycin phosphotransfease gene as reporter genes is used for transformation.
-
by Agrobacterium-mediated transformation. Spores of Marchantia polymorpha are germinated and grown into immature thalli. The 7-day-old immature thalli are co-cultivated with Agrobacterium and transferred directly to selective M51C agar medium after washing. Incubation of immature thalli with Agrobacterium harboring the binary plasmid pIG121Hm lead to the formation of hygromycin-resistant plantlets, whereas Agrobacterium carrying no binary plasmid did not. Hygromycin-resistant thalli with rhizoids became distinct 10 days after transfer to the selection agar medium, whereas hygromycin-sensitive plantlets developed into chlorotic cell clumps. To avoid chimerism of hygromycin-resistant thalli, isogenic lines are obtained from gemmae which arise asexually from single initial cells in cupules, and used for further analysis.
-
Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate plasmid pGV2260::pSSJ1 carried the hygromycin phosphotransferase and beta-glucuronidase genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase gene under maize ubiquitin promoter.
-
DNA sequence determination and analysis, overexpression of His-tagged enzyme in strain DH5alpha in inclusion bodies
-
Embryogenic culture of Alfalfa plant is transformed using Agrobacterium tumefaciens containing the super binary plasmid pToK233 that encodes for the neomycin phosphotransferase II, hygromycin phosphotransferase and glucuronidase genes in order to design an antibiotic resistant line.
-
expression in 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)
-
gene hph, subcloning in strain DH5alpha, expression in Neurospora crassa 74-OR23-IVA and al-1,mcm
-
gene hph, subcloning in strain JM109, expression of wild-type and mutant enzymes in Thermus thermophilus strain HB27, establishment of the host-vector system using the hpt gene as a selective marker
-
hph5 gene is introduced into Thermus thermophilus on a plasmid of eight copies. HPH and HPH5 are PCR amplified and Escherichia coli strain BL21 (DE3) is transformed with the plasmids and cultivated.
-
hyg-gene introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene is inserted into the BamHI site of pBR322 and then cloned in Escherichia coli phosphorylating activity is not detected. When the hyg gene is inserted into either the unique PstI site of the pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity is observed
-
hygromycin B resistance gene is cloned in pBR322, recombinant plasmids pKC241 pKC222 carrying the resistance
-
in Colletotrichum falcatum and Colletotrichum acutatum. Agrobacterium tumefaciens-mediated transformation (ATMT) is used by using the vector pBHt2 that contains a t-DNA harboring the hygromycin B resistance gen (hygromycin phosphotransferase, hph) in the backbone of pCAMBIA1300.
-
Linearized DNA of plasmid pV2 bearing the hygromycin B phosphotransferase (hph) gene is inserted into chromosomes of wild strain T23 resulting in improved capability of degrading organophosphate pesticide dichlorvos. Transformation is confirmed by PCR and Southern blot analysis. 76% of transformants show improved dichlorvos degradation ability as compared to the parent strain T23
-
Physical parameters for transient transformation are optimized using the UidA gene encoding beta-glucuronidase as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker
-
Producing many transgenic cyclamen plants. Agrobacterium tumefaciens strain LBA4404 harbors the binary vector plasmid pIG121Hm, which contains selectable marker genes for hygromycin phosphotransferase and neomycin phosphotransferase is used for transformation.
-
Reproducible procedure for transformation of shoot apices and regeneration of transgenic plants for two indica rice cultivars. Shoot apex explants are transformed by cocultivation with Agrobacterium tumefaciens strain EHA 101 harbouring the binary plasmid pRIT1. Vector contains an improved hygromycin phosphotransferase gene for hygromycin resistance driven by actin 1 promoter and the reporter gene beta-glucuronidase intron controlled by CaMV 35S promoter.
-
subcloning in strain DH5alpha, overexpression of non-tagged enzyme in strain BL21(DE3) in inclusion bodies, method optimization, expression in transgenic rice plants
-
The hpt coding region is obtained by a XhoI digestion of the vector pCAMBIA1380 and is cloned into the SalI-cut pBluescript KSII. The EcoRV-XhoI fragment of the hpt coding sequence is ligated with the SmaI-XhoI fragment of pHAG to obtain the intermediate plasmid pHyII, so that the hpt coding region is flanked by the 2.3 kb ASA2 promoter at the 5'end and the Arabidopsis actin2 terminator at the 3'end. Finally, the SacI-SacII fragment of pHyII is ligated into the SacI-SacII-cut pBluescript KSII to complete the transformation vector pXZIII-8.
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D20G/A118V/S225P/Q226L/T246A
-
site-directed mutagenesis, mutant gene hpt5, mutant enzyme is stable at up to 67C in contrary to the wild-type enzyme
S52T
-
site-directed mutagenesis, the mutation does not confer thermostability at 55C to the mutant enzyme
W238C
-
site-directed mutagenesis, the mutation does not confer thermostability at 55C to the mutant enzyme
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from inclusion bodies after overexpression in Escherichia coli, solubilization by 0.3% sarcosine, renaturation by dilution and dialysis, method optimization
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
molecular biology