Information on EC 3.4.22.B79 - nsP2 protease

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.22.B79
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
nsP2 protease
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
the enzymes processes the alphavirus nonstructural polyprotein (nsP1234). The enzyme from Venezuelan equine encephalitis virus shwos a preferens for Gly or Als in position P1', Ala or Cys in P1, and Gly in P2
show the reaction diagram
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
MBPNusG-His6 p3/p4 + H2O
?
show the reaction diagram
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-
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?
P123 polyprotein + H2O
?
show the reaction diagram
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-
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?
P23 polyprotein + H2O
?
show the reaction diagram
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-
-
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?
Semliki forest virus p1/p2 + H2O
?
show the reaction diagram
Semliki forest virus p3/p4 + H2O
?
show the reaction diagram
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-
?
Sindbis virus core protein + H2O
Hydrolyzed Sindbis core protein
show the reaction diagram
thioredoxin fusion protein + H2O
?
show the reaction diagram
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fusion protein is cleaved with greater efficiency by nsp2pro than the original MBP-NusG-His6 substrate with the shorter linker
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?
Trx34 + H2O
?
show the reaction diagram
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?
Venezuelan equine encephalitis virus p1/p2 + H2O
?
show the reaction diagram
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?
Venezuelan equine encephalitis virus p3/p4 + H2O
?
show the reaction diagram
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?
XJ-160 virus-specific protein + H2O
?
show the reaction diagram
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?
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
XJ-160 virus-specific protein + H2O
?
show the reaction diagram
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?
additional information
?
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enzyme is critically involved in development of cytopathic effect. Cytotoxic effect of enzyme is due to its ability to cause transcriptional shutoff
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
EDTA
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inhibition of nsp2pro at or above 2 mM EDTA
glycerol
additional information
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.58 - 1.2
Semliki forest virus p1/p2
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1.27
Semliki forest virus p3/p4
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0.25
Trx34
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wild-type, at 28C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 0.043
Semliki forest virus p1/p2
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0.352
Semliki forest virus p3/p4
Semliki forest virus
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 30
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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SIN/nsP2GFP/8-specific fusion protein is present in the cytoplasm in large complexes. SIN/nsP2GFP/472 also present
Manually annotated by BRENDA team
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very little amounts of SIN/nsP2GFP/8-specific fusion protein. SIN/nsP2GFP/472 also present
Manually annotated by BRENDA team
additional information
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SIN/nsP2GFP/8-specific fusion protein is present in the cytoplasm in large complexes, and very little is found in the nuclei, nsP2/GFP of SIN/nsP2GFP/472 is distributed both in the cytoplasm and in the nucleus
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Manually annotated by BRENDA team
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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cytotoxic functions of of enzyme are determined by the integrity of the carboxy-terminal peptide rather than its protease activity and depend on the presence of protein in a free form
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
nonstructural proteins nsP1-4 are produced as a single polyprotein, and processing of the polyprotein occurs in a highly regulated manner. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible. The nsP2 protease active site is over 40 A away from the P2/3 cleavage site, supporting a trans cleavage mechanism
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
homology model of nsP2 protein based on the crystal structure of the nsP2 protein of Venezuelan equine encephalitis virus and proposal of the the pharmacophore features that must be present in an inhibitor of nsP2 protease
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molecular dynamics simulation and virtual screening of inhibitors based on PDB entry 3TRK leads to identification of top hit compounds, together with the five potential binding pockets of the nsP2 protease. Pocket 4 in the N-terminal domain of the nsP2 protease has been identified as the active site by the presence of catalytic residues, Cys1013 and His1083
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hanging drop vapor diffusion method, using 12%(w/v) PEG 8000, 10%(v/v) ethylene glycol, 100 mM HEPES pH 7.5
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structure of nonstructural protein P23 in a precleavage form. The proteins form an extensive interface and nsP3 creates a ring structure that encircles nsP2. The P2/3 cleavage site is located at the base of a narrow cleft and is not readily accessible. The nsP2 protease active site is over 40 A away from the P2/3 cleavage site, supporting a trans cleavage mechanism. nsP3 contains a previously uncharacterized protein fold with a zinc-coordination site. Known mutations in nsP2 that result in formation of noncytopathic viruses or a temperature sensitive phenotype cluster at the nsP2/nsP3 interface
at 2.45 A resolution. Belongs to space group P212121. N-terminal domain (Asp468 to Asn603) contains the catalytic dyad formed by Cys477 and His546, organized in a protein fold. C-terminal domain (Arg604 to Ser793) contributes to substrate recognition and regulates the structure of the substrate binding cleft
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combination of molecular dynamics simulations with structural studies. The catalytic mechanism of the nsP2 protease appears similar to the papain-like cysteine proteases, with the conserved catalytic dyad forming a thiolate-imidazolium ion pair in the nsP2-activated state. Substrate binding likely stabilizes this ion pair. Protease residues His510, Ser511, His546, and Lys706 are critical for cleavage site recognition. Structural determinants of substrate specificity that recognize features common to all three cleavage sites within the viral polyprotein are strongly conserved among alphaviruses. Contacts between S2/P2 and S3/P3 residues illustrate preserved binding motifs such as the ubiquitous P2 glycine interaction with Trp547 and hydrogen bonding between P2 and His510
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-70C, 20 mM HEPES buffer, pH 7.4, 200 mM NaCl, 20% glycerol, 0.1% Tween 20, 1 mM DTT, 1 mM EDTA, 12 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by gel filtration, more than 99% pure
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fusion proteins purified by immobilized metal affinity chromatography, to homogeneity
GST-glutathione affinity column chromatography
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Pro39 mutants purified by metal affinity chromatography
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA coding for nsP2pro (residues Met457-Cys794) amplified and cloned into vector pETBlue1 T7. Expression in Tuner DE3 (pLacI) Escherichia coli cells
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expressed in BHK-21 cells
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expressed initially as HisMBP fusion proteins in Escherichia coli BL21(DE3) CodonPlus-RIL cells
GFP-encoding DNA fragment cloned into the nsP2 gene by using a transposon-based approach. The entire library of the recombinant nsP2/GFP genes transferred into wild-type Sindbis virus Toto1101 genome. SIN/nsP2GFP/8 and SIN/nsP2GFP/472 expressed in BHK-21 cells
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GFP-encoding DNA fragment randomly cloned into the nsP2 gene. Entire library of the recombinant nsP2/GFP genes transferred into wild-type SINV Toto1101 genome. BHK-21 cells infected with SIN/nsP2GFP/8 and with both packaged replicons and SIN/nsP2GFP/472 virus
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Pro39 mutants expressed in Escherichia coli BL21
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the gene that encodes a double-mutant IBV nsp2 N-terminal domain (residues 9-393 of the polyprotein, with mutations Q132L and L270F) is expressed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A662T
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temperature-sensitive in vivo and in vitro
C478A
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inactive
G577R
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temperature-sensitive in vivo and in vitro
H548A
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inactive
M781T
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temperature-sensitive in vivo and in vitro
N600D
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significantly impaired activity. Slower growth rate and smaller bacterial mass as well as a reduced expression level of the corresponding soluble Pro39. Results in preparates with lower degree of purity and shows a tendency to aggregate
N605D
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significantly impaired activity. Slower growth rate and smaller bacterial mass as well as a reduced expression level of the corresponding soluble Pro39. Results in preparates with lower degree of purity and shows a tendency to aggregate
W549A
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inactive
C481G
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
C481R
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
C481S
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
C525R
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
C525S
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mutant is active and processed normally
G8063V
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mutation blocks P2/3 cleavage during P123 processing
H558A
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
H558Q
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
H558Y
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
H619A
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mutant is active and processed normally
H701A
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mutant is active and processed normally
H709A
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mutant is active and processed normally
H709R
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
H709Y
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mutant is active and processed normally
K173E
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mutation has any influence on the activity of Sindbis virus nsp2pro
N561D
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mutant shows some activity, with normal processing
N561S
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mutant shows some activity, with normal processing
N609D
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mutant is active, almost no processing
N609S
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mutant is active, with reduced processing
N614D
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mutant shows enhanced processing, and is lethal
N614S
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mutant shows some activity, with normal processing
N693S
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mutant shows some activity, and is lethal
P726A
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the mutant exhibits similar growth characteristics to the wild type BR-XJ160 in cultured cells, including cytopathic effects, plaque morphology and growth kinetics
P726L
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the mutant shows no cytopathic effects or plaques after six passages through BHK-21 cells, although expression of XJ-160 virus-specific protein is detectable, the mutant produces no lethality or morbidity in suckling mice
P726S
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the mutant shows reduced growth capacity in cultured cells and mouse brain, and intermediate neurovirulence
P726V
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the mutant exhibits similar growth characteristics to the wild type BR-XJ160 in cultured cells, including cytopathic effects, plaque morphology and growth kinetics
S535T
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mutant is active and processed normally
Y559A
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mutation in C-terminal protease domain, abolishes catalytic activity, and is lethal
G17V
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
N545D
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
Q471L
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
T5A
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
T5I
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
V3A
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mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
medicine
additional information
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nsP2/GFP is capable of efficient functioning in Sindbis virus replication complexes that can synthesize RNA and, ultimately, produce virus at a level comparable to that of wild-type Sindbis virus. GFP inserted into nsP2 is accessible to specific antibodies and capable of functioning as an efficient tag for the isolation of protein complexes formed during Sindbis virus replication