Information on EC 3.4.22.45 - helper-component proteinase

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

EC NUMBER
COMMENTARY hide
3.4.22.45
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RECOMMENDED NAME
GeneOntology No.
helper-component proteinase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
hydrolyses a Gly-/-Gly bond at its own C-terminus, commonly in the sequence -Tyr-Xaa-Val-Gly-/-Gly, in the processing of the potyviral polyprotein
show the reaction diagram
endopeptidase; Known from many potyviruses. The helper component-proteinase of the tobacco etch virus is a multifunctional protein with several known activities: the N-terminal region is required for aphid transmission and efficient genome amplification, the central region is required for long-distance movement in plants, and the C-terminal domain has cysteine endopeptidase activity. Type example of peptidase family C6
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of peptide bond
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CAS REGISTRY NUMBER
COMMENTARY hide
124566-20-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
potyvirus, clover yellow vein virus no. 30, induction of lethal necrosis in the broad bean Vicia faba
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Manually annotated by BRENDA team
i.e. CMV
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Manually annotated by BRENDA team
no activity in cassava brown streak virus
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Manually annotated by BRENDA team
no activity in Squash vein yellowing virus
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Manually annotated by BRENDA team
type-W
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Manually annotated by BRENDA team
genome polyprotein; PPV
SwissProt
Manually annotated by BRENDA team
potato virus A PVA-B11
polyprotein; PVA
UniProt
Manually annotated by BRENDA team
potato virus Y N
genome polyprotein, includes helper component proteinase HC-pro; N strain
SwissProt
Manually annotated by BRENDA team
potato virus Y Nevski
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Manually annotated by BRENDA team
potato virus Y PVY-Nevski
polyprotein; PVY
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
polyprotein; TEV
UniProt
Manually annotated by BRENDA team
Tobacco vein mottling potyvirus
ssRNA positive-strand virus, no DNA stage
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Manually annotated by BRENDA team
strain California, ZYMV
SwissProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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amino acids essential for the proteolytic activity of ZYMV HC-Pro are distinct from those of the tobacco etch virus HC-Pro, although the amino acid sequences in the proteolytic active domain are conserved among potyviruses
malfunction
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
HC-Pro + H2O
?
show the reaction diagram
potyvirus helper component + H2O
?
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
HC-Pro + H2O
?
show the reaction diagram
potyvirus helper component + H2O
?
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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kinetic of interactions between recombinant His-tagged enzyme and 20S proteasome
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
sRNA binding assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the RNA binding ability is temperature-sensitive
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Turnip mosaic virus (strain Japanese)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
126000
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recombinant MBP:HA-HC-Pro:GFP
138000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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nested deletion and domain mapping for enzyme analysis, overview
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
two-dimensional crystal of the recombinant proteins are successfully grown on Ni2+-chelating lipid monolayxers. Comparison of projection maps of negatively stained crystals reveal that the enzyme is composed of two domains separated by a flexible constriction
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as glutathione S-transferase-tag fusion protein
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HC-Pro protein, wild-type and mutant protein, gel filtration
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His-tagged wild-type enzyme and an N-terminal deletion mutant
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HisTrap column chromatography
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mutant and wild-type of HC-Pro proteins, SDS-PAGE
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recombinant dimeric His-tagged HC-Pro proteinase from Escherichia coli
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recombinant enzyme from transfected plants and from Pichia pastoris GS115, purification of TEV virion particles
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recombinant HC-Pro protein of potato virus Y and its deletion mutants
recombinant HC-Pro protein, SDS-PAGE
recombinant His-tagged enzyme from LMV in plant leaves by nickel affinity chromatography and gel filtration, recombinant Strep-tagged enzyme by ammonum sulfate fractionation and gel filtration
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recombinant MBP-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by affinity purification using amylose magnetic beads
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recombinant N-terminally His6-tagged or MBP-fused wild-type enzyme and MBP-fused mutant HC-ProFINK protein from Escherichia coli strain BL21 (DE3) by affinity chromatography
recombinant protein
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
14 isolates of turnip mosaic virus, RNA extraction, reverse transctiption, amplification using RT-PCR, cloned into pGEM-T vector for sequence analysis and phylogenetic analysis. HC-Pro genes comprise 1374 nucleotides encoding a polypeptide of 458 amino acids
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42 isolates of plum pox virus (different types of strains. plum pox virus-D, plum pox virus-M, plum pox virus-Rec). Single-strand conformation polymorphism analysis and low-stringency single specific primer PCR analysis are performed on the HC-Pro region of the genome for genotyping
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as P1/HC-Pro, expressed in Nicotiana benthamiana
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binary constructs for overexpression of helper component proteinase HC-Pro in Tobacco etch virus generated, overexpression in Nicotina benthamiana via infiltration with Agrobacterium tumefaciens shown, binary vector pBin19-GFP used, pHannibal vector used for GFP-silencing inverted-repeat constructs
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expressed in Arabidopsis
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expressed in Escherichia coli
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expressed in Escherichia coli and Nicotiana tabacum; expressed in Escherichia coli and Nicotiana tabacum
expressed in Escherichia coli JM109 (DE3) cells
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expressed in Escherichia coli, transformation into Agrobacterium tumefaciens C58C1 by using the binary plant expression vector pBIN61Sa
expressed in Nicotiana benthamiana
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expression in immature Glycine max cotyledonary explants, co-transfection and co-culture with Agrobacterium tumefaciens strain KYRT1 transfected with a hygromycin phosphotransferase gene, the enzyme enhances recorvery of somatic embryos of Glycine max, effects on gene expression, overview
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expression of HCPro with YN fused to the HCpro N terminus in Nicotiana benthamiana, and translation initiation factors eIF(iso)4E (Tiso4Eb and Piso4Eb) and eIF4E (T4Ea and P4Eb) of tobacco (T) and potato (P) with YC fused to the N- or C-terminus. Leaves are infiltrated with pairs of Agrobacterium strains expressing tester proteins tagged with the opposite halves of YFP
expression of HCPro with YN fused to the HCpro N terminus in Nicotiana benthamiana, and translation initiation factors eIF(iso)4E (Tiso4Eb and Piso4Eb) and eIF4E (T4Ea and P4Eb) of tobacco (T) and potato (P) with YC fused to the N- or C-terminus. Leaves are infiltrated with pairs of Agrobacterium strains expressing tester proteins tagged with the opposite halves of YFP. Interactions are negative in the YTHS, an artificial system in which protein interactions are tested in the nucleus
expression of His6-tagged or MBP-fused wild-type enzyme and of MBP-fused mutant HC-ProFINK protein in Escherichia coli strain BL21 (DE3)
expression of recombinant His-tagged or Strep-tagged enzyme in LMV in plant leaves
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expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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fragment of pS81-SA containing the HC-Pro protein coding region used for generation of substitution mutations, ligation into pGEM5zf+ and transformation into Escherichia coli DH5a, 250 PCR-generated clones screened for mutations by single-strand conformation polymorphism SSCP analysis
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full-length coding sequence of HC-Pro of potato virus Y fused to the GAL4 DNA-binding domain, cloned into pGBKT7 via BamHI/PstI digestion to form pGBKT7-HC-Pro, different plasmid constructs generated, overview of domains and deletion mutants of HC-Pro given, transformation into Saccharomyces cerevisiae after subcloning into pGBKT7
functional expression of wild-type MBP:HA-HC-Pro:GFP and truncated mutant MBP:HA-HC-Pro:GFP N1, i.e. enzyme tagged with HA, GFP, and maltose-binding protein, in Escherichia coli leads to autoproteolytic cleavage of the green fluorescent protein, GFP
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genetic organization, enzyme replacements, overview
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HC-Pro is indispensable for Papaya ringspot virus infection in zucchini, N-terminus of HC-Pro is involved in systemic infection of PRSV
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HC-Pro is involved in virulence on Rsv1-genotype soybean (Rsv1 is a single dominant resistance gene), influence of single-point mutations on virulence analyzed
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HC-Pro suppresses the RNA silencing induced by sense RNA and dsRNA
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in vitro translation of transcripts in rabbit reticulocyte lysate or wheat germ extract, expression in Escherichia coli as His-tagged protein
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mutations in HC-Pro for analysing virulence on soybean, HC-Pro complementation of viral protein P3 is essential for virulence on Rsv1-genotype soybean (Rsv1 is a single dominant resistance gene)
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nucleotide and amino acid sequence determination, coding region of 1152 nucleotides, expression of wild-type and deletion mutants in recombinant Wheat streak mosaic virus in wheat
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plasmid pTEV7DA with an infectious TEV clone used as source of wild-type virus and template for site-directed mutagenesis. Wild-type and mutants are cloned into pBIN61 vector, electroporation of Agrobacterium tumefaciens for transient expression assays and quantification of suppression activity.
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recombinant expression using Potato virus X, PVX, in Nicotiana benthamiana, expression induces the pathogenicity of the Potato virus X manifesting a necrosis in plant and plant death
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small and large scale functional expression in Pichia pastoris strain GS115 of the gene fused into the Saccharomyces cerevisiae alpha-mating factor secretory peptide coding region, plant transfection using the Myus persicae aphid vector
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subcloned into pALTER vector for generation of substitution mutations by site-directed mutagenesis
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used in yeast two-hybrid assay
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used in yeast two-hybrid system, expressed in Escherichia coli, expressed in onion tissue (fusion protein)
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wild-type and mutant protein, expressed in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D193Y
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mutants of helper component protease HC-Pro of clover yellow vein virus, generated by site-directed mutagenesis or by PCR-based primer extension mutagenesis, revertant of D193Y also created, single mutant alone does not induce lethal necrosis
I33
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mutants of helper component protease HC-Pro of clover yellow vein virus generated by site-directed mutagenesis or by PCR-based primer extension mutagenesis
R51I
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mutants of helper component protease HC-Pro of clover yellow vein virus generated by site-directed mutagenesis or by PCR-based primer extension mutagenesis
T27I
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mutants of helper component protease HC-Pro of clover yellow vein virus generated by site-directed mutagenesis or by PCR-based primer extension mutagenesis, revertant of T27I also created, single mutant alone does not induce lethal necrosis but retains ability to induce necrotic symptoms
T27I/D193Y
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double mutant
C344A/H417A
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the mutant shows abolished self-cleavage
K52E/K52A
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the mutations disrupt interaction between the enzyme and aphids
L134H
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the mutant shows abolished RNA-silencing suppression
P310A/T311A
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the mutations disrupt interaction between the enzyme and capsid protein
RK182,4AA
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the mutant shows abolished RNA-silencing suppression
RK246,7AA
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the mutant shows abolished RNA-silencing suppression
RRH234,5,6AAA
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the mutant shows abolished self-cleavage
I225V
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defective in aphid transmission activity
K50E
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defective in aphid transmission activity
D446A
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mutation does not abolish in vitro interaction between helper-component-proteinase and coat protein
E450A
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mutation does not abolish in vitro interaction between helper-component-proteinase and coat protein
H429A
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mutation does not abolish in vitro interaction between helper-component-proteinase and coat protein
K432A
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mutation does not abolish in vitro interaction between helper-component-proteinase and coat protein
K452A
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mutation does not abolish in vitro interaction between helper-component-proteinase and coat protein
R455A
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mutation abolishes in vitro interaction between helper-component-proteinase and coat protein
T310A
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mutation abolishes in vitro interaction between helper-component-proteinase and coat protein
A387E
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
C390W
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
C649S
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active site mutant, no activity
C694S
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mutant is proteolytically active and capable of cell-to-cell and long-distance movements
D421N
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
D715E
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mutant is proteolytically active and capable of cell-to-cell and long-distance movements
E273A
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
E299A/D300A
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RNA silencing suppressor activity reduced compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
E360A/D361A
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RNA silencing suppressor activity reduced compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows mild etching
E443K
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RNA silencing suppressor activity increased compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
E452D
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
H722S
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active site mutant, no activity
I11L
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RNA silencing suppressor activity increased compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
I442M
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
K309N
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
K454T
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RNA silencing suppressor activity increased compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
N193Y
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
N194D
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
N200S
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RNA silencing suppressor activity increased compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
P311L
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
Q265H
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
R127G
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
R165G
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
R240A/K241A/H242A
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
R247A/K248A
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
S610T
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mutant is proteolytically active and capable of cell-to-cell and long-distance movements
V135A
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no RNA silencing suppressor activity, Nicotiana benthamiana plant inoculated with infectious transcript shows no etching
V192A
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RNA silencing suppressor activity reduced compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows mild etching
V355L
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
V419A
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
Y234H
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
Y344S
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RNA silencing suppressor activity similar to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows mild etching
Y423H
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RNA silencing suppressor activity increased compared to wild-type, Nicotiana benthamiana plant inoculated with infectious transcript shows etching
C291S
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inactive in aphid transmission
C305S
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active in aphid transmission, similar to wild-type
K307E
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active in aphid transmission
K307H
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inactive in aphid transmission
K307Q
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inactive in aphid transmission
K307R
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active in aphid transmission
K51E
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defective in aphid transmission activity
C16A
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substitution mutants introduced into amino-proximal region of helper component protease HC-Pro, vector transmission abolished
C16AC20A
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double mutant generated, vector transmission abolished
C20A
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no effects on vector transmission
C20R
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no effects on vector transmission, randomized substitution
C46A
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vector transmission abolished
C49A
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vector transmission abolished
C49AC46A
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double mutant generated, vector transmission abolished
K7N
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no effects on vector transmission, randomized substitution
N19I
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no effects on vector transmission, randomized substitution
R45K
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no effects on vector transmission, randomized substitution
D458G
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no effect on zucchini yellow mosaic virus symptoms
D506Y
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very mild symptoms of zucchini yellow mosaic virus infection
E396N
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affects symptom severity and viral pathogenicity
F205L
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affects symptom severity and viral pathogenicity
R180I
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affects symptom severity and viral pathogenicity
R180I/E396N
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induces transient leaf mottling, affects symptom severity and viral pathogenicity
R180I/F205L/E396N
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the mutant is completely defective in its capacity to block miRNA regulation
Y309A
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defective in aphid transmission activity
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture