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Information on EC 3.4.22.B79 - nsP2 protease and Organism(s) Venezuelan equine encephalitis virus and UniProt Accession P27282

for references in articles please use BRENDA:EC3.4.22.B79
preliminary BRENDA-supplied EC number
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EC Tree
     3 Hydrolases
         3.4 Acting on peptide bonds (peptidases)
             3.4.22 Cysteine endopeptidases
                3.4.22.B79 nsP2 protease
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Venezuelan equine encephalitis virus
UNIPROT: P27282
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The taxonomic range for the selected organisms is: Venezuelan equine encephalitis virus
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
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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
Synonyms
nonstructural protein 2, nonstructural polyprotein, nsp2 protease, nsp2 protein, nsp2pro, ns polyprotein, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
Semliki forest virus p1/p2 + H2O
?
show the reaction diagram
-
-
-
?
Venezuelan equine encephalitis virus p1/p2 + H2O
?
show the reaction diagram
-
-
-
?
Venezuelan equine encephalitis virus p3/p4 + H2O
?
show the reaction diagram
-
-
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
E64d
inhibitor binds beneath a beta-hairpin at the interface of the SAM MTase and protease domains
glycerol
activity is reduced by approximately 20fold in the presence of 5% glycerol
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.58
Semliki forest virus p1/p2
-
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016
Semliki forest virus p1/p2
-
-
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
-
nsP2 is either directly or indirectly involved in the RNA encapsidation process
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
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
free enzyme and adduct with inhibitor E64d
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
K480A
mutation leads to a 9fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys residue
K706A
SAM MTase domain mutation, increases Km 4.5fold to 500 microM
N475A
N545A
mutation within the beta-hairpin, lightly but not significantly increases kcat and Km
R662A
mutation leads to 16fold decreases in kcat/Km
R662K
mutation leads to 16fold decreases in kcat/Km
S701A
mutation leads to a 17fold increase in Km
G17V
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
N545D
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
Q471L
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
T5A
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
T5I
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
V3A
-
mutation causes an increase in infectous viral titer, variants become cytopathic for both BHK-21 and Vero cells
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
activity decreases dramatically at temperatures above 30°C
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
by gel filtration, more than 99% pure
fusion proteins purified by immobilized metal affinity chromatography, to homogeneity
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
expressed initially as HisMBP fusion proteins in Escherichia coli BL21(DE3) CodonPlus-RIL cells
expression in Escherichia coli
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
drug development
alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases
medicine
the nsp2pro N-terminal domain is a novel cysteine protease fold. the C-terminal domain displays structural similarity to S-adenosyl-L-methionine-dependent RNA methyltransferases. This structure will significantly aid drug discovery and development efforts to combat Venezuelan equine encephalitis alphavirus and related viruses
analysis
assay for quantitative measurement of Nsp2 protease activity based on a substrate fusion protein consisting of eGFP and Gaussia luciferase linked together by a small peptide containing a Nsp2 protease cleavage sequence. The expression of the substrate protein in cells along with recombinant Nsp2 protease results in cleavage of the substrate protein resulting in extracellular release of free Gaussia luciferase
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Russo, A.T.; White, M.A.; Watowich, S.J.
The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease
Structure
14
1449-1458
2006
Venezuelan equine encephalitis virus (P27282)
Manually annotated by BRENDA team
Zhang, D.; Toezser, J.; Waugh, D.S.
Molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses
Protein Expr. Purif.
64
89-97
2009
Semliki forest virus, Sindbis virus, Venezuelan equine encephalitis virus (P27282), Venezuelan equine encephalitis virus
Manually annotated by BRENDA team
Russo, A.T.; Malmstrom, R.D.; White, M.A.; Watowich, S.J.
Structural basis for substrate specificity of alphavirus nsP2 proteases
J. Mol. Graph. Model.
29
46-53
2010
Venezuelan equine encephalitis virus (P27282), Venezuelan equine encephalitis virus
Manually annotated by BRENDA team
Kim, D.Y.; Atasheva, S.; Frolova, E.I.; Frolov, I.
Venezuelan equine encephalitis virus nsP2 protein regulates packaging of the viral genome into infectious virions
J. Virol.
87
4202-4213
2013
Venezuelan equine encephalitis virus
Manually annotated by BRENDA team
Hu, X.; Compton, J.R.; Leary, D.H.; Olson, M.A.; Lee, M.S.; Cheung, J.; Ye, W.; Ferrer, M.; Southall, N.; Jadhav, A.; Morazzani, E.M.; Glass, P.J.; Marugan, J.; Legler, P.M.
Kinetic, mutational, and structural studies of the Venezuelan equine encephalitis virus nonstructural protein 2 cysteine protease
Biochemistry
55
3007-3019
2016
Venezuelan equine encephalitis virus (P27282), Venezuelan equine encephalitis virus, Venezuelan equine encephalitis virus Trinidad donkey (P27282)
Manually annotated by BRENDA team
Compton, J.R.; Mickey, M.J.; Hu, X.; Marugan, J.J.; Legler, P.M.
Mutation of Asn-475 in the Venezuelan equine encephalitis virus nsP2 cysteine protease leads to a self-inhibited state
Biochemistry
56
6221-6230
2017
Venezuelan equine encephalitis virus (P27282), Venezuelan equine encephalitis virus Trinidad donkey (P27282)
Manually annotated by BRENDA team
Campos-Gomez, J.; Ahmad, F.; Rodriguez, E.; Saeed, M.F.
A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease
Virology
496
77-89
2016
Venezuelan equine encephalitis virus (Q9WJC7), Venezuelan equine encephalitis virus, Venezuelan equine encephalitis virus Mena II (Q9WJC7)
Manually annotated by BRENDA team