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Information on EC 3.2.2.22 - rRNA N-glycosylase and Organism(s) Phytolacca americana and UniProt Accession Q40772
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3.2.2.22 rRNA N-glycosylaseIUBMB Comments
Ricin A-chain and related toxins show this activity. Naked rRNA is attacked more slowly than rRNA in intact ribosomes. Naked rRNA from Escherichia coli is cleaved at a corresponding position.
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The taxonomic range for the selected organisms is: Phytolacca americana
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
pd-l1, ricin, shiga toxin, saporin, abrin, ricinus communis agglutinin, gelonin, ricin a chain, trichosanthin, ribosome-inactivating proteins, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
gelonin
-
-
-
-
Mirabilis antiviral protein
-
-
-
-
momorcochin-S
-
-
-
-
nigrin b
-
-
-
-
PAP
PAP-S
pokeweed anti-viral protein
pokeweed antiviral protein
ribosomal ribonucleate N-glycosidase
-
-
-
-
ricin
-
-
-
-
RNA N-glycosidase
-
-
-
-
rRNA N-glycosidase
-
-
-
-
saporins
-
-
-
-
type I ribosome-inactivating protein
type I RIP
PAP
-
recombinantly combined with karasurin-A (KRN) of Trichosanthes kirilowii
type I ribosome-inactivating protein
-
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of the N-glycosylic bond at A-4324 in 28S rRNA from rat ribosomes
catalytic mechanism
-
hydrolysis of the N-glycosylic bond at A-4324 in 28S rRNA from rat ribosomes
anti-viral mechanism of type 1 RIP pokeweed anti-viral protein PAP
-
hydrolysis of the N-glycosylic bond at A-4324 in 28S rRNA from rat ribosomes
N70 of PAP is required for depurination catalytic activity, N70 facilitates depurination in trans
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of N-glycosyl bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
rRNA N-glycohydrolase
Ricin A-chain and related toxins show this activity. Naked rRNA is attacked more slowly than rRNA in intact ribosomes. Naked rRNA from Escherichia coli is cleaved at a corresponding position.
CAS REGISTRY NUMBER
COMMENTARY
113756-12-0
-
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
large RNA + H2O
?
removal of a single adenine
-
-
?
2'-anthraniloyl 7-methylguanosine triphosphate + H2O
?
-
depurination downstream of cap structure, 2.4-fold enhanced binding to m7GTP upon the addition of translation initiation factor G4 (elFiso4G)
-
-
?
25S rNA + H2O
?
-
-
enzyme depurinates the sarcin/ricin loop when the A-site of the ribosomal peptidyl-transferase center is unoccupied
-
?
28Sdelta rRNA + H2O
?
-
depurination of the A51 residue of Trypanosoma cruzi 28Sdelta rRNA, the C-terminal end of ribosomal P proteins is not required for full activity
-
?
8-vinyl-2'-deoxyadenosine containing 10-mer stem-tetraloop RNA + H2O
8-vinyladenine + ?
-
-
slow
-
?
large RNA + H2O
?
removal of a single adenine
-
-
?
brome mosaic virus RNA + H2O
?
-
depurination of the viral RNA, anti-viral activity of PAP by inhibition of accumulation of viral RNA and viral proliferation in barley protoplasts, overview
-
-
?
RNA + H2O
?
-
the enzyme is active on viral RNA, e.g. from Human immunodeficiency virus-1 and Brome mosaic virus, RNA accession mechanism via interaction with translation initiation factors 4G and iso4G, overview
-
-
?
RNA + H2O
?
-
the enzyme is active on viral RNA, e.g. from Human immunodeficiency virus-1 and Brome mosaic virus
-
-
?
RNA + H2O
?
-
ribosomes from rat liver (eukaryotic) and Escherichia coli JM109 (prokaryotic), 37°C, 25 mM Tris-HCl buffer, pH 7.6 with 50 mM KCl and 5 mM MgCl2
-
-
?
rRNA + H2O
?
-
removes G4323 from rabbit reticulocyte, tobacco and yeast ribosomes
-
-
?
rRNA + H2O
?
-
depurination of the sarcin-ricin loop of eukaryotic large rRNA
-
-
?
rRNA + H2O
?
PAP causes translation arrest and is cytotoxic in targeted cells
-
-
?
rRNA + H2O
?
pokeweed antiviral protein binds to ribosomes and depurinates a specific adenine in the highly conserved alpha-sarcin/ricin loop of the large subunit rRNA
-
-
?
rRNA + H2O
?
-
a total of 20 and 40 microg of ribosome inactivating protein on 1 square cm discs of Physeolus vulgaris leaves, monitoring of weight gain (day 4 and 10), survival rate, DNA lesions, and oxidative status as catalase and superoxide dismutase activities and lipidic peroxidation in third instar butterfly larvae of Noctuidae Anticarsia gemmatalis and Spodoptera frugiperda
-
-
?
supercoiled double-stranded DNA + H2O
nicked and linear forms of DNA
-
-
-
-
?
supercoiled double-stranded DNA + H2O
nicked and linear forms of DNA
-
highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycosylase activities, DNA modifications other than depurination may be attributed to contamination with nucleases, gelonin, momordin I, PAP-S, and saporin-S6 do not cleave DNA strands, the only base released is adenine, it may be concluded that polynucleotide:adenosine glycosidase is the enzymatic activity on DNA of these, and possibly all, ribosome-inactivating proteins
-
-
?
additional information
?
-
-
anti-viral activity of PAP in CBA mice infected with lymphocytic choriomeningitis virus, LCMV strain WE54, inoculated by intracerebral injections, overview, anti-viral activity against HIV-1 in human peripheral blood mononuclear cells infected with strain HIV-1 HLTVIIIB, IC50p24 is 14 nM
-
-
?
additional information
?
-
-
PAP shows high antiviral activity against both plant and animal viruses, mechanism study, viral particles are not attacked directly, the ribosome inactivation is not essential for antiviral effectivity, PAP acts as immunotoxin, overview
-
-
?
additional information
?
-
-
anti-viral activity of PAP in vitro against lymphocytic choriomeningitis virus, LCMV, and against HIV-1
-
-
?
additional information
?
-
-
PAP does not show rRNA depurination activity
-
-
?
additional information
?
-
-
the enzyme interacts with translation initiation factors 4G and iso4G, binding and interaction study with wild-type and mutant factors, overview, PAP binds to thr m7G cap structure
-
-
?
additional information
?
-
-
the enzyme shows polynucleotide:adenosine glycosidase, PAGase, activity
-
-
?
additional information
?
-
-
depurination of rRNA, N-glycosidase activity, oxidative and genotoxic action on butterfly third instar larvae Anticarsia gemmatalis (soybean caterpillar) and Spodoptera frugiperda (fall armyworm) of the lepidopteran family Noctuidae
-
-
?
additional information
?
-
-
PAP shows RNA N-glycosidase activity towards eukaryotic and prokaryotic ribosomes, karasurin-A only towards eukaryotic ribosomes, removal of specific adenine from alpha-sarcin/ricin loop of the large rRNA, substrate specificity (eukaryotic/prokaryotic) determined by C-terminus residues 209-225
-
-
?
additional information
?
-
-
removal of adenine from rRNA, inactivating ribosomal function in translation
-
-
?
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
brome mosaic virus RNA + H2O
?
-
depurination of the viral RNA, anti-viral activity of PAP by inhibition of accumulation of viral RNA and viral proliferation in barley protoplasts, overview
-
-
?
RNA + H2O
?
-
the enzyme is active on viral RNA, e.g. from Human immunodeficiency virus-1 and Brome mosaic virus, RNA accession mechanism via interaction with translation initiation factors 4G and iso4G, overview
-
-
?
rRNA + H2O
?
-
depurination of the sarcin-ricin loop of eukaryotic large rRNA
-
-
?
rRNA + H2O
?
PAP causes translation arrest and is cytotoxic in targeted cells
-
-
?
additional information
?
-
-
anti-viral activity of PAP in CBA mice infected with lymphocytic choriomeningitis virus, LCMV strain WE54, inoculated by intracerebral injections, overview, anti-viral activity against HIV-1 in human peripheral blood mononuclear cells infected with strain HIV-1 HLTVIIIB, IC50p24 is 14 nM
-
-
?
additional information
?
-
-
PAP shows high antiviral activity against both plant and animal viruses, mechanism study, viral particles are not attacked directly, the ribosome inactivation is not essential for antiviral effectivity, PAP acts as immunotoxin, overview
-
-
?
additional information
?
-
-
depurination of rRNA, N-glycosidase activity, oxidative and genotoxic action on butterfly third instar larvae Anticarsia gemmatalis (soybean caterpillar) and Spodoptera frugiperda (fall armyworm) of the lepidopteran family Noctuidae
-
-
?
additional information
?
-
-
PAP shows RNA N-glycosidase activity towards eukaryotic and prokaryotic ribosomes, karasurin-A only towards eukaryotic ribosomes, removal of specific adenine from alpha-sarcin/ricin loop of the large rRNA, substrate specificity (eukaryotic/prokaryotic) determined by C-terminus residues 209-225
-
-
?
additional information
?
-
-
removal of adenine from rRNA, inactivating ribosomal function in translation
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
puromycin
-
prior incubation of ribosomes with puromycin inhibits both the binding and depurination by the enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
-
PAP-S: increase in cells that show DNA damage in both species, reduced weight gain of Spodoptera frugiperda (not Anticarsia gemmatalis) on day 4 with 40 microg, increased weight gain with 20 microg in Spodoptera frugiperda, not significant increase in weight gain in Anticarsia gemmatalis fed with 40 microg, reduced weight in Anticarsia gemmatalis and Spodoptera frugiperda at day 10 with 40 microg, 57.1% mortality in Anticarsia gemmatalis, in Anticarsia gemmatalis 30-40% reduced level of lipid peroxidation with 40 microg, in Spodoptera frugiperda superoxide dismutase inhibition to 20% and catalase activity increase with 40 microg
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
PAP in spring leaf and probably during the whole plant life
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
additional information
the enzyme is synthesized as inactive precursor protein, accumulated in the endoplasmic reticulum associated to membranes, and needs to cleaved to the mature active form in the endoplasmic reticulum of the targeted cell, the mature form is retro-translocated from the ER to the cytosol where it is accumulated and escapes degradation, residues E176 and N70 are essential for catalytic activity
-
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). RIP2_PHYAM
310
0
34694
Swiss-Prot
Secretory Pathway (Reliability: 1)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
proteolytic modification
proteolytic modification
the enzyme is synthesized as inactive precursor protein and needs to cleaved to the mature active form in the endoplasmic reticulum of the targeted cell, the mature form is retro-translocated from the ER to the cytosol where it escapes degradation
proteolytic modification
-
enzyme is synthesized in pokeweed plants as a 313-amino-acid precursor with a 22-amino-acid N-terminal signal sequence and a 29-amino-acid C-terminal extension
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E176V
site-directed mutagenesis, inactive mutant showing no cytotoxicity
G75D
-
no maturation of precursor form, localization exclusivelly in the membrane fraction
L252stop
-
accumulation of the truncated protein primarily in the membrane fraction, at the lumenal side of microsomal membranes
N253stop
-
reduced accumulation of the protein in the cytosol, appearance of a slightly larger form in the membrane fraction
N70A
site-directed mutagenesis, the mutation delays the enzyme depurination activity on ribosomes and the onset of translation arrest, N70A PAP shows highly reduced cytotoxicity and destabilizes its own mRNA, but binds to cap and blocks cap-dependent translation
V73E
-
no maturation of precursor form, reduced stability, localized to the lumenal side of microsomal membranes
additional information
additional information
-
expression of PAP in transgenic plants, e.g. Nicotiana tabacum, confers resistance to potato virus X and fungus Rhizoconia solani
additional information
-
chimera proteins of 3 domains of pokeweed antiviral protein (PAP) and karasurin-A (N-terminal 1-74, central 75-177, C-terminal 178-262), C-terminal end determines determines specificity to eukaryotic or eukaryotic and prokaryotic ribosomes, PAP-region 209-225 is required for activity towards prokaryotic ribosomes
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant PAP-II from Escherichia coli strain JM101 by an alkaline method
cell centrifugation, washing, lysozyme application, sonication, centrifugation, supernatants applied to GSTrap FF column, elution with sonication buffer and 10 mM reduced glutathione, desalted, concentrated and glutathione S-transferase (GST) cleaved off with PreScission Protease
-
homogenized frozen leaves filtered through cheesecloth, centrifuged, saturated with ammonium sulfate to 60%, stirred, all at 4 degrees Celsius, centrifuged, pellet resuspended in 20 mM Tris-HCl, pH 7.5 with 1 mM beta-mercaptoethanol, and 0.2 mM EDTA, dialyzed against buffer, centrifuged, loaded onto HiTrap Q FF column, bound proteins loaded onto HiTrap SP FF column, eluted with NaCl gradient, PAP elutes at about 250 mM NaCl
-
native PAP from spring leaves in four steps, ammonium sulfate fractionation, anion and cation exchange chromatography, and dialysis, to homogeneity
-
recombinant His-tagged wild-type and mutant PAPs from Escherichia coli strain BL21(DE3) by m7G affinity chromatography
recombinant PAP from Escherichia coli strain JM101 by an alkaline method
recombinant PAP-R from Escherichia coli strain JM101 by an alkaline method
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination of PAP-II, expression in Escherichia coli strain JM101
DNA and amino acid sequence determination of PAP, expression in Escherichia coli strain JM101
DNA and amino acid sequence determination of PAP-R, expression in Escherichia coli strain JM101
DNA and amino acid sequence determination of PAP-S, expression in Escherichia coli strain JM101
expression in Saccharomyces cerevisiae strain mak8-1 which is more resistant to the enzyme in vivo due to higher affinity for aminoacyl-tRNA
-
expression of His-tagged wild-type and mutant PAPs in Escherichia coli strain BL21(DE3)
expression of wild-type PAP in yeast, and of mutant PAPs in Escherichia coli
-
plasmid pMAL-c2 to amplify pokeweed antiviral protein and karasurin-A by PCR, expression in Escherichia coli XL1-Blue cells with expression plasmid pGEX-6P-1
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Endo, Y.; Tsurugi, K.; Lambert, J.M.
The site of action of six different ribosome-inactivating proteins from plants on eukaryotic ribosomes: the RNA N-glycosidase activity of the proteins
Biochem. Biophys. Res. Commun.
150
1032-1036
1988
Suregada multiflora, Momordica charantia, Phytolacca americana, Saponaria officinalis
Barbieri, L.; Valbonesi, P.; Righi, F.; Zuccheri, G.; Monti, F.; Gorini, P.; Samori, B.; Stirpe, F.
Polynucleotide:Adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA
J. Biochem.
128
883-889
2000
Suregada multiflora, Momordica charantia, Phytolacca americana, Saponaria officinalis
Peumans, W.J.; Hao, Q.; Van Damme, E.J.
Ribosome-inactivating proteins from plants: more than RNA N-glycosidases?
FASEB J.
15
1493-1506
2001
Abrus precatorius, Ricinus communis, Eranthis hyemalis, Suregada multiflora, Hordeum vulgare, Momordica charantia, no activity in Arabidopsis thaliana, Phytolacca americana, Phytolacca insularis, Saponaria officinalis, Trichosanthes kirilowii, Trichosanthes sp.
Zhao, Y.; Wang, X.; Zhou, G.; Li, H.
A pokeweed antiviral protein gene in roots of Phytolacca americana
Acta Virol.
48
131-132
2004
Phytolacca americana (P93444), Phytolacca americana (Q40772), Phytolacca americana (Q53YN2), Phytolacca americana (Q8RYA4), Phytolacca americana
Parikh, B.A.; Baykal, U.; Di, R.; Tumer, N.E.
Evidence for retro-translocation of pokeweed antiviral protein from endoplasmic reticulum into cytosol and separation of its activity on ribosomes from its activity on capped RNA
Biochemistry
44
2478-2490
2005
Phytolacca americana (P10297)
Hartley, M.R.; Lord, J.M.
Cytotoxic ribosome-inactivating lectins from plants
Biochim. Biophys. Acta
1701
1-14
2004
Abrus precatorius, Cucurbita pepo, Dianthus caryophyllus, Escherichia coli, Mirabilis jalapa, Momordica charantia, Phytolacca americana, Sambucus nigra, Saponaria officinalis, Shigella dysenteriae, Trichosanthes kirilowii, Triticum aestivum, Viscum album, Adenia volkensii, Sambucus ebulus, Ricinus communis (P02879)
Uckun, F.M.; Rustamova, L.; Vassilev, A.O.; Tibbles, H.E.; Petkevich, A.S.
CNS activity of pokeweed anti-viral protein (PAP) in mice infected with lymphocytic choriomeningitis virus (LCMV)
BMC Infect. Dis.
5
9
2005
Phytolacca americana
Picard, D.; Kao, C.C.; Hudak, K.A.
Pokeweed antiviral protein inhibits brome mosaic virus replication in plant cells
J. Biol. Chem.
280
20069-20075
2005
Phytolacca americana
Wang, M.; Hudak, K.A.
A novel interaction of pokeweed antiviral protein with translation initiation factors 4G and iso4G: a potential indirect mechanism to access viral RNAs
Nucleic Acids Res.
34
1174-1181
2006
Phytolacca americana
Stirpe, F.
Ribosome-inactivating proteins
Toxicon
44
371-383
2004
Abrus precatorius, Ricinus communis, Suregada multiflora, Hordeum vulgare, Phytolacca americana, Sambucus nigra, Saponaria officinalis, Shigella dysenteriae, Trichosanthes kirilowii, Viscum album, Zea mays, Phoradendron californicum, Adenia volkensii, Adenia digitata, Mirabilis expansa
Ayub, M.J.; Smulski, C.R.; Ma, K.W.; Levin, M.J.; Shaw, P.C.; Wong, K.B.
The C-terminal end of P proteins mediates ribosome inactivation by trichosanthin but does not affect the pokeweed antiviral protein activity
Biochem. Biophys. Res. Commun.
369
314-319
2008
Trichosanthes kirilowii (P09989), Phytolacca americana (P10297)
Roday, S.; Saen-oon, S.; Schramm, V.L.
Vinyldeoxyadenosine in a sarcin-ricin RNA loop and its binding to ricin toxin A-chain
Biochemistry
46
6169-6182
2007
Phytolacca americana, Ricinus communis (P02879)
Baykal, U.; Tumer, N.E.
The C-terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity
Plant J.
49
995-1007
2007
Phytolacca americana
Mansouri, S.; Nourollahzadeh, E.; Hudak, K.A.
Pokeweed antiviral protein depurinates the sarcin/ricin loop of the rRNA prior to binding of aminoacyl-tRNA to the ribosomal A-site
RNA
12
1683-1692
2006
Phytolacca americana
Baldwin, A.E.; Khan, M.A.; Tumer, N.E.; Goss, D.J.; Friedland, D.E.
Characterization of pokeweed antiviral protein binding to mRNA cap analogs: competition with nucleotides and enhancement by translation initiation factor iso4G
Biochim. Biophys. Acta
1789
109-116
2009
Phytolacca americana
Bertholdo-Vargas, L.R.; Martins, J.N.; Bordin, D.; Salvador, M.; Schafer, A.E.; Barros, N.M.; Barbieri, L.; Stirpe, F.; Carlini, C.R.
Type 1 ribosome-inactivating proteins - entomotoxic, oxidative and genotoxic action on Anticarsia gemmatalis (Huebner) and Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)
J. Insect Physiol.
55
51-58
2009
Suregada multiflora, Momordica charantia, Phytolacca americana, Saponaria officinalis, Silene chalcedonica
Nagasawa, Y.; Fujii, K.; Yoshikawa, T.; Kobayashi, Y.; Kondo, T.
Pokeweed antiviral protein region Gly209-Lys225 is critical for RNA N-glycosidase activity of the prokaryotic ribosome
Phytochemistry
69
1653-1660
2008
Phytolacca americana, Trichosanthes kirilowii (P24478)
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