Information on EC 3.4.22.66 - calicivirin

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The expected taxonomic range for this enzyme is: ssRNA positive-strand viruses, no DNA stage

EC NUMBER
COMMENTARY hide
3.4.22.66
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RECOMMENDED NAME
GeneOntology No.
calicivirin
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
endopeptidase with a preference for cleavage when the P1 position is occupied by Glu-/- and the P1' position is occupied by Gly-/-
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
-
-
-
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CAS REGISTRY NUMBER
COMMENTARY hide
218925-73-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
hepatitis A virus
HAV
-
-
Manually annotated by BRENDA team
MD145 virus
Noro virus strain MD145
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-
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
norovirus MD145
strain MD145
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-
Manually annotated by BRENDA team
norovirus MD145-12
strain MD145-12
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-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
SaV
-
-
Manually annotated by BRENDA team
SARS-CoV
-
-
Manually annotated by BRENDA team
TGEV
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Dabcyl-GLRTQSFS-Edans + H2O
Dabcyl-GLRTQ + SFS-Edans
show the reaction diagram
hepatitis A virus
-
-
-
-
?
dabcyl-KTSAVLQSGFRKME-EDANS + H2O
?
show the reaction diagram
-
-
-
-
?
Dabcyl-KTSAVLQSGFRKME-EDANS + H2O
Dabcyl-KTSAVLQ + SGFRKME-EDANS
show the reaction diagram
Edans-APAKQLLN-Dabcyl + H2O
Edans-APAKQ + LLN-Dabcyl
show the reaction diagram
-
-
-
-
?
Edans-DFHLQGP-Dabcyl + H2O
Edans-DFHLQ + GP-Dabcyl
show the reaction diagram
Glu-Thr-Thr-Leu-Glu-Gly-Gly-Asp
Glu-Thr-Thr-Leu-Glu + Gly-Gly-Asp
show the reaction diagram
-
model of an oligopeptide bound to CVP
-
-
?
His3Cd-GST-protein + H2O
?
show the reaction diagram
-
-
-
-
?
Murine norovirus nonstructural polyprotein + H2O
?
show the reaction diagram
-
clevage map
-
-
?
norovirus polyprotein + H2O
hydrolyzed norovirus polyprotein
show the reaction diagram
-
-
-
-
?
ORF1 polyprotein + H2O
nonstructural proteins
show the reaction diagram
-
-
-
-
?
ORF1 polyprotein + H2O
precursor comprised of both the proteinase and polymerase + mature polymerase
show the reaction diagram
ORF1 polyprotein + H2O
six nonstructural proteins for replication
show the reaction diagram
-
-
-
?
poly(A)-binding protein
?
show the reaction diagram
Poly(A)-binding protein + H2O
?
show the reaction diagram
cleavage separates the C-terminal domain of the substrate that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain
-
-
?
Southampton viral polyprotein precursor p113 + H2O
p48 N-terminal + p41 helicase + 22000 Da 3A-like protein + 16000 Da Vpg + 19000 Da 3C-like viral protease + 57000 Da 3D polymerase
show the reaction diagram
-
cleavage sites are Q399-/-G400, Q762-/-G763, E961-/-G962, E1099-/-A1100 and E1280-/-G1281
-
?
additional information
?
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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
norovirus polyprotein + H2O
hydrolyzed norovirus polyprotein
show the reaction diagram
-
-
-
-
?
Southampton viral polyprotein precursor p113 + H2O
p48 N-terminal + p41 helicase + 22000 Da 3A-like protein + 16000 Da Vpg + 19000 Da 3C-like viral protease + 57000 Da 3D polymerase
show the reaction diagram
-
cleavage sites are Q399-/-G400, Q762-/-G763, E961-/-G962, E1099-/-A1100 and E1280-/-G1281
-
?
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2S)-2-([N-[(benzyloxy)carbonyl]-L-leucyl]amino)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonic acid
2-mercaptoethanol
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20% inhibited by 0.01 mM, 40% inhibited by 5 mM
4-(2-aminoethyl)-benzene-sulfonyl fluoride
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irreversible inhibition, soluble stable nontoxic alternative to phenylmethylsulfonyl fluoride
Hg2+
-
5 mM completely inhibits
K+
-
inhibited by 100 mM
L-Leu-Gly-Gly
-
-
methyl methanethiosulfonate
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almost complete inhibition
N-ethylmaleimide
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almost complete inhibition
N-Phenylmaleimide
-
almost complete inhibition
N2-[(benzyloxy)carbonyl]-N-[(2S)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl]-L-leucinamide
N2-[(benzyloxy)carbonyl]-N-[(2S)-3,4-dioxo-1-(2-oxopyrrolidin-3-yl)-4-(propan-2-ylamino)butan-2-yl]-L-leucinamide
Na+
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83% inhibited by 100 mM
p-chloromercuribenzenesulfonate
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27% inhibited by 0.01 mM
p-chloromercuribenzoic acid
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82% inhibited by 0.01 mM
phenyl methylsulfonylfluoride
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20% inhibition by 0.01 mM
phenylmethylsulfonyl fluoride
-
-
rupintrivir
zinc N-ethyl-N-phenyldithiocarbamate
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-
Zn2+
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5 mM completely inhibits
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Glu 54
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active role in proteolysis
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IPTG
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induction of gene expression
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00049 - 0.05
(2S)-2-([N-[(benzyloxy)carbonyl]-L-leucyl]amino)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonic acid
0.00061 - 0.05
N2-[(benzyloxy)carbonyl]-N-[(2S)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl]-L-leucinamide
0.00055 - 0.0426
N2-[(benzyloxy)carbonyl]-N-[(2S)-3,4-dioxo-1-(2-oxopyrrolidin-3-yl)-4-(propan-2-ylamino)butan-2-yl]-L-leucinamide
0.0007 - 0.05
rupintrivir
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
hepatitis A virus
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assay at
8.62
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100% enzyme activity
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.22 - 9.08
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 48
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-
PDB
SCOP
CATH
ORGANISM
UNIPROT
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Norwalk virus (strain GI/Human/United States/Norwalk/1968)
Sapporo virus (strain Human/United Kingdom/Manchester/1993)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
19000
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calculation from nucleotide sequence
20600
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calculated from ProHis protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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size exclusion chromatography results
homodimer
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x-ray crystallography
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion grown at 20C, at 2.8 A resolution
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sitting drop vapor diffusion method, using 18% (w/v) PEG 6000, 10% (v/v) DMSO, 14% methyl-2,4-pentanediol, and 0.12 M Tris-HCl, pH 8.5
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by hanging drop vapor diffusion or sitting drop vapor diffusion at room temperature, to 1.5 and 2.2 A resolution
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purified recombinant enzyme free and bound to inhibitor (2S)-2-([N-[(benzyloxy)carbonyl]-L-leucyl]amino)-1-hydroxy-3-(2-oxopyrrolidin-3-yl)propane-1-sulfonic acid, X-ray diffraction structure determination and analysis at 1.50 and 1.65 A resolution, respectively, molecular replacement
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purified recombinant enzyme bound to inhibitor GC376, X-ray diffraction structure determination and analysis at 2.25 A resolution, molecular replacement
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by nickel affinity chromatography or size exclusion chromatography
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GSTrap FF column chromatography
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Ni-NTA column chromatography and HiTrap 26/10 QFF column chroamtography
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purified over chitin beads and by gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into pET41 Ek/LIC and pET46 Ek/LIC and expressed in Escherichia coli BL21 (DE3)
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expressed in Escherichia coli BL21 cells
-
expressed in Escherichia coli BL21-CodonPlus(DE3)-RIPL cells
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expression in Escherichia coli
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expression in Escherichia coli BL21 (DE3)
expression in HEK293 cells
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expression in HeLa cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C139A
-
cysteine residue responsible for hydrolysis
E54L
-
the E54L mutant protease is a substrate-specificity mutant and requires large hydrophobic amino acid residues at both P4 and P2 positions of the substrate, it cleaves the sequence 133LSFE/AP between the 3B and 3C regions of norovirus polyprotein, but does not cleaves the sequence 198ATSE/GK between the 3A and 3B
C122S
-
site-directed mutagenesis, the mutant is affected in its catalytic activity of proteolytic processing
C139S
-
site-directed mutagenesis, the mutant is affected in its catalytic activity of proteolytic processing
E54A
-
Glu at the active centre
C104S
-
site-directed mutagenesis, the mutant is affected in its catalytic activity of proteolytic processing
C116S
-
site-directed mutagenesis, the mutant is affected in its catalytic activity of proteolytic processing
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine