Information on EC 3.4.21.115 - infectious pancreatic necrosis birnavirus Vp4 peptidase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.21.115
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RECOMMENDED NAME
GeneOntology No.
infectious pancreatic necrosis birnavirus Vp4 peptidase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cleaves the (Ser/Thr)-Xaa-Ala-/-(Ser/Ala)-Gly motif in the polyprotein NH2-pVP2-VP4-VP3-COOH of infectious pancreatic necrosis virus at the pVP2-VP4 and VP4-VP3 junctions
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
CAS REGISTRY NUMBER
COMMENTARY hide
330588-95-9
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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Manually annotated by BRENDA team
MABV strain Y-6, isolated from yellowtail Seriola quinqueradiata with ascites
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Manually annotated by BRENDA team
residue 619-830
UniProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
NH2-pVP2-X-VP4-VP3-COOH + H2O
pVP2 + peptide X + VP4 + VP3
show the reaction diagram
VP4 is encoded as part of the 1114-residue long segment A polyprotein NH2-pVP2-X-VP4-VP3-COOH. It cleaves after amino acid residue positions 512, 618, and 830 to yield capsid precursor protein pVP2, peptide X, VP4 itself, and ribonucleoprotein VP3. Additional cleavage sites were found at the C-terminal side of amino acids 451, 492, and 499 within the pVP2 region
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?
polyprotein of BSNV + H2O
pVP2 + VP3 + VP4
show the reaction diagram
polyprotein of IPNV + H2O
pVP2 + VP3 + VP4
show the reaction diagram
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
polyprotein of BSNV + H2O
pVP2 + VP3 + VP4
show the reaction diagram
polyprotein of IPNV + H2O
pVP2 + VP3 + VP4
show the reaction diagram
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the viral NS protease is active and cleaves the polyprotein when it is expressed in Escherichia coli
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
first identification of an acyl-enzyme complex for a Ser/Lys dyad protease, catalytic mechanism and substrate recognition indicated
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.9
calculated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20500
recombinant protein VP4_524-716-K674A (VP4hex), 194 residues long, theoretical isoelectric point of 4.3 estimated
21700
recombinant protein VP4_514-716-K674A (VP4tri), 204 residues long, theoretical isoelectric point of4.5 estimated
24745
x * 24745, calculated from amino acid sequence
240000
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SDS-PAGE, single band of VP4, same size of VP4 in all analyzed cell lines
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 24745, calculated from amino acid sequence
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a truncated variant with 18 residues removed from the C-terminal
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two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries, refinement to 2.2 A resolution reveals the acyl-enzyme complex formed with an internal VP4 cleavage site, substrate binding pockets S1, S3, S5, and S6 identified, conserved overall structure of VP4 determined by three-dimensional alignments
VP4 with the C-terminus binding into its own active site forming an intramolecular (cis) acyl-enzyme complex, hanging drop vapor diffusion method, using 21% (w/v) PEG8000 and 0.55 M ammonium sulfate
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate fractionation and size-exclusion chromatography
eluted from SDS-PAGE gel
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gel filtration, native and recombinant proteins
Ni-NTA column chromatography, SP-Sepharose column chromatography, and Sephacryl S-100 gel filtration
two chromatography steps to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli strain BL21-DE3 using the pET-41b vector
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expressed in Escherichia coli Tuner (DE3) cells
expressed in Escherichia coli Tuner (DE3) cells using the pET-24a vector, two recombinant constructs generated by site-directed mutagenesis of the IPNV VP4 protease analyzed
expression in Escherichia coli
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expression in Escherichia coli JM107
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wild-type IPNA and truncated forms, expressed in Escherichia coli
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
P774Z
18 residues removed from the C-terminus
D537Q
mutant with wild-type activity
D585I
mutant with wild-type activity
D595L
mutant with wild-type activity
D601S
mutant with wild-type activity
D644I
mutant with wild-type activity
D660G/D661S
mutant with wild-type activity
D672N
mutant with wild-type activity
D693L
reduced activity for the VP4-VP3 junction
H547S
reduced activity for the VP4-VP3 junction
H697L
reduced activity for the VP4-VP3 junction
H704S
mutant with wild-type activity
K674A
two recombinant constructs VP4_524-716-K674A and VP4_514-716-K674A generated by site-directed mutagenesis
S633C
40% reduction of VP2-VP4 cleavage, 80% reduction of VP4-VP3 cleavage
H681F
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amino acid substitutions generated in VP4 of MABV by site-directed mutagenesis
I543G
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amino acid substitutions generated in VP4 of MABV by site-directed mutagenesis
K674D
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amino acid substitutions generated in VP4 of MABV by site-directed mutagenesis
S633P
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amino acid substitutions generated in VP4 of MABV by site-directed mutagenesis
V686Q
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amino acid substitutions generated in VP4 of MABV by site-directed mutagenesis