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Information on EC 2.7.1.163 - hygromycin B 4-O-kinase and Organism(s) Escherichia coli and UniProt Accession P00557
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2.7.1.163 hygromycin B 4-O-kinaseIUBMB Comments
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.
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Word Map
- 2.7.1.163
- agrobacterium
- molecular biology
-
agrobacterium-mediated
-
explants
- biotechnology
- tumefaciens
-
tumefaciens-mediated
-
hygromycin-resistant
- acetosyringone
-
plantlets
-
cocultivation
-
microalgal
-
embryogenic
-
pcambia
- majus
- nptii
-
protocorms
-
calluses
-
orchid
-
reintegration
The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Reaction Schemes
Synonyms
hptii, hygromycin-phosphotransferase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
Hph5
hygromycin phosphotransferase
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
88361-67-5
cf. EC 2.7.1.119
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + destomycin A
ADP + 4-O-phosphodestomycin A
-
1-N-methyl isomer of hygromycin B
-
-
?
ATP + destomycin B
ADP + 4-O-phosphodestomycin B
-
1-N-methyl-4',4"-epi hygromycin B
-
-
?
ATP + hygromycin B
ADP + 4-O-phosphohygromycin B
-
phosphorylated product procures hygromcin B resistance
-
-
ir
ATP + hygromycin B2
ADP + 4-O-phosphohygromycin B2
-
pseudodisaccharide of D-talose and hyosamine
-
-
?
hygromycin B + ATP
7''-O-phosphohygromycin B + ADP
-
resulting in a loss of cell-kill antibiotic activity of hygromycin B
-
-
r
hygromycin B + ATP
4-O-phosphohygromycin B + ADP
-
Growth of Colletotrichum falcatum is completely inhibited at 50 microg/ml, whereas in Colletotrichum acutatum, a concentration of 300 microg/ml is most effective in inhibiting mycelial growth
-
-
?
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
-
-
?
additional information
?
-
-
the analogs of destomycin A and B, A23444 (N-demethyl hygromycin B) and SS-56C (N-demethyl-2-hydroxy hygromycin B) with modifications in the cyclitol ring moiety, are phosphoryted by the enzyme
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
ATP + hygromycin B
ADP + 4-O-phosphohygromycin B
-
phosphorylated product procures hygromcin B resistance
-
-
ir
hygromycin B + ATP
7''-O-phosphohygromycin B + ADP
-
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
-
-
?
hygromycin B + ATP
4-O-phosphohygromycin B + ADP
-
Growth of Colletotrichum falcatum is completely inhibited at 50 microg/ml, whereas in Colletotrichum acutatum, a concentration of 300 microg/ml is most effective in inhibiting mycelial growth
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
107471 counts/minute/25 microl sample extract, hygromycin as substrate
additional information
-
14328 counts/minute/25 microl sample extract, A23444 as substrate
additional information
-
15508 counts/minute/25 microl sample extract, SS-56C as substrate
additional information
-
24483 counts/minute/25 microl sample extract, destomycin A as substrate
additional information
-
35387 counts/minute/25 microl sample extract, strain BE1065 containing plasmid pKC222
additional information
-
50051 counts/minute/25 microl sample extract, strain BE1098 containing plasmid pKC241
additional information
-
5986 counts/minute/25 microl sample extract, hygromycin b2 as substrate
additional information
-
8428 counts/minute/25 microl sample extract, destomycin B as substrate
additional information
-
hygromycin B is an aminocyclitol antibiotic with broad-spectrum activity against both procaryotic and eucaryotic cells
additional information
-
No activity is found in the plasmid-free DH1 strain.
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
hph gene detected in two unapproved transgenic rice lines contaminating vermicelli products
UniProt
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
-
Agrobacterium tumefaciens strain LBA4404 harbors the binary vector plasmid pIG121Hm containing hpt gene from Escherichia coli
-
strains W677, BE783, BE827, DH1, BE1065, BE1092, and BE1098 are used
additional information
hph gene descending from the plasmid pJR225 is detected in food (rice noodles)
additional information
-
Immature thalli of Marchantia developing from spores are used for Agrobacterium tumefaciens infection
additional information
-
Seeds cultivar DCS-9 (Jyoti) of Ricinus communis L. are used for gun-mediated gene transfer. The cultivars 48-1 and VP-1 are also used for particle bombardement.
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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using a thermostable mutant and the hanging-drop vapour-diffusion method at 20°C. Thermostable proteins crystallize with less difficulty than wild-type proteins
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A118V
D20G
D20G/A118V/S225P/Q226L/T246A
-
site-directed mutagenesis, mutant gene hpt5, mutant enzyme is stable at up to 67°C in contrary to the wild-type enzyme
Q226L
S225P
S52T
-
site-directed mutagenesis, the mutation does not confer thermostability at 55°C to the mutant enzyme
T246A
W238C
-
site-directed mutagenesis, the mutation does not confer thermostability at 55°C to the mutant enzyme
additional information
A118V
-
site-directed mutagenesis, the mutation is involved in conferring thermostability at 55°C to the mutant enzyme
D20G
-
site-directed mutagenesis, the mutation is involved in conferring thermostability at 55°C to the mutant enzyme
Q226L
-
site-directed mutagenesis, the mutation is involved in conferring thermostability at 55°C to the mutant enzyme
S225P
-
site-directed mutagenesis, the mutation is involved in conferring thermostability at 55°C to the mutant enzyme
T246A
-
site-directed mutagenesis, the mutation is involved in conferring thermostability at 55°C to the mutant enzyme
additional information
-
construction of a hygromycin-sensitive Neurospora crassa mutant heterokaryon strain by crossing of a wild-type strain and a stable hygromycin transformant, the enzyme can be reversibly inactivated, overview
additional information
-
construction of transgenic Oryza sativa plants expressing the enzyme in leaves, roots, and seeds
additional information
-
mutant enzyme containing all mutations shows an increased thermostability of approximately 16°C K in vivo compared with the wild-type protein
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
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 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.
-
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 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.
-
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.
-
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.
-
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
LITERATURE
biotechnology
molecular biology
biotechnology
-
the enzyme can be used as selective marker gene product in production of transgenic plants
biotechnology
-
the enzyme is widely used as selective marker gene product in production of engineered crops, e.g. rice
biotechnology
-
resistance against hyromycin B mediated by transformation of the hph gene
molecular biology
-
the enzyme can be used as selective marker gene product in production of transgenic plants
molecular biology
-
the mutant gene hph5 gene can be used as a selection marker in the host-vector system of Thermus thermophilus either on plasmid or by genome integration
molecular biology
-
gene is used as selectable marker, mediates hygromycin resistence
molecular biology
-
gene sequence is used as selectable marker, mediates hygromycin resistance
molecular biology
-
hpt gene is used as selectable marker, mediates hygromycin resistance
molecular biology
-
resistance against hyromycin B mediated by transformation of the hph gene
molecular biology
-
resistance against hyromycin B mediated by transformation of the hph gene, a selectable marker gen.
molecular biology
-
selectable marker (SM) genes, essential to select transformed cells from a large population of untransformed cells
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Yang, L.C.; Zhu, Z.; Yang, X.G.
Purification and immunity analysis of recombinant 6His-HPT protein expressed in E. coli
Biomed. Environ. Sci.
16
149-156
2003
Escherichia coli
Dev, K.; Maheshwari, R.
Silencing of hygromycin phosphotransferase (hph) gene during sexual cycle and its reversible inactivation in heterokaryon of Neurospora crassa
Curr. Microbiol.
47
220-225
2003
Escherichia coli
Nakamura, A.; Takakura, Y.; Kobayashi, H.; Hoshino, T.
In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus
J. Biosci. Bioeng.
100
158-163
2005
Escherichia coli
Zhuo, Q.; Piao, J.H.; Wang, R.; Yang, X.G.
Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies
Protein Expr. Purif.
41
53-60
2005
Escherichia coli
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
Acta Crystallogr. Sect. F
63
685-688
2007
Escherichia coli
Rao, R.N.; Allen, N.E.; Hobbs, J.N.; Alborn, W.E.; Kirst, H.A.; Paschal, J.W.
Genetic and enzymatic basis of hygromycin B resistance in Escherichia coli
Antimicrob. Agents Chemother.
24
689-695
1983
Escherichia coli
Nakamura, A.; Takakura, Y.; Sugimoto, N.; Takaya, N.; Shiraki, K.; Hoshino, T.
Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase
Biosci. Biotechnol. Biochem.
72
2467-2471
2008
Escherichia coli
Akiyama, H.; Sasaki, N.; Sakata, K.; Ohmori, K.; Toyota, A.; Kikuchi, Y.; Watanabe, T.; Furui, S.; Kitta, K.; Maitani, T.
Indicated detection of two unapproved transgenic rice lines contaminating vermicelli products
J. Agric. Food Chem.
55
5942-5947
2007
Escherichia coli (P00557)
Maruthachalam, K.; Nair, V.; Rho, H.S.; Choi, J.; Kim, S.; Lee, Y.H.
Agrobacterium tumefaciens-mediated transformation in Colletotrichum falcatum and C. acutatum
J. Microbiol. Biotechnol.
18
234-241
2008
Escherichia coli
Platisa, J.; Veljovic-Jovanovic, S.; Kukavica, B.; Vinterhalter, B.; Smigocki, A.; Ninkovic, S.
Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)
J. Plant Physiol.
165
895-900
2008
Escherichia coli
Terakawa, T.; Yamamura, T.; Murayama, T.
Improvement of regeneration and transformation systems for Cyclamen persicum using somatic embryo culture
Plant Biotechnol.
25
77-80
2008
Escherichia coli
-
Ishizaki, K.; Chiyoda, S.; Yamato, K.T.; Kohchi, T.
Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology
Plant Cell Physiol.
49
1084-1091
2008
Escherichia coli
Arockiasamy, S.; Ignacimuthu, S.
Regeneration of transgenic plants from two indica rice (Oryza sativa L.) cultivars using shoot apex explants
Plant Cell Rep.
26
1745-1753
2007
Escherichia coli
Sailaja, M.; Tarakeswari, M.; Sujatha, M.
Stable genetic transformation of castor (Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds
Plant Cell Rep.
27
1509-1519
2008
Escherichia coli
Sripriya, R.; Raghupathy, V.; Veluthambi, K.
Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain
Plant Cell Rep.
27
1635-1644
2008
Escherichia coli
Zernova, O.; Zhong, W.; Zhang, X.H.; Widholm, J.
Tissue culture specificity of the tobacco ASA2 promoter driving hpt as a selectable marker for soybean transformation selection
Plant Cell Rep.
27
1705-1711
2008
Escherichia coli
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