Information on EC 3.4.21.B30 - UmuD protein

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.4.21.B30
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
UmuD protein
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
involved in UV protection and mutation. Essential for induced (or SOS) mutagenesis. May modify the DNA replication machinery to allow bypass synthesis across a damaged template
show the reaction diagram
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CAS REGISTRY NUMBER
COMMENTARY hide
98059-81-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene umuDC
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Manually annotated by BRENDA team
gene umuD
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Manually annotated by BRENDA team
strain FC40
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
malfunction
metabolism
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competition between enzyme UmuD and ssDNA for DNA polymerase III alpha binding is a distinct mechanism for polymerase exchange
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
UmuD + H2O
?
show the reaction diagram
UmuD undergoes cleavage upon interaction with a RecA/ssDNA complex
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?
UmuD + H2O
UmuD' + ?
show the reaction diagram
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auto-cleavage of UmuD to UmuD'
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-
?
UmuD2 + H2O
UmuD2' + ?
show the reaction diagram
UmuDAb + H2O
UmuDAb' + ?
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
UmuD + H2O
UmuD' + ?
show the reaction diagram
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auto-cleavage of UmuD to UmuD'
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-
?
UmuD2 + H2O
UmuD2' + ?
show the reaction diagram
UmuDAb + H2O
UmuDAb' + ?
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
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required for RecA-mediated cleavage
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DNA polymerase III
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UmuD interacts with the beta subunit of DNA polymerase III
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RecA
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
14000
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UmuD', SDS-PAGE
15000
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2 * 15000, recombinant enzyme UmuD, SDS-PAGE
15063
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2 * 15063
15300
calculated from amino acid sequence
15500
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2 * 15500, predicted from amino acid sequence
17000
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UmuD, SDS-PAGE
23000
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SDS-PAGE
24000
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x * 24000, recombinant enzyme variant UmuDAb, SDS-PAGE
30000
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dimer, calculated from nucleic acid sequence
31000
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predicted from amino acid sequence
40000
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dimer, SDS-PAGE
72000
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cross-linked tetramers, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
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UmuD and UmuD’ rapidly form heterodimers in vitro
homodimer
tetramer
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two dimers form a tetramer, wild-type and V34C mutant, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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UmuD2 and UmuD'2 each have a melting temperature of 60°C
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
highly purified UmuD is specifically degraded in vitro by Lon protease in an ATP-dependent manner, UmuD' is insensitive to proteolysis by Lon
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highly purified UmuD/D' heterodimer is degraded in the presence of ATP, ClpP and ClpX, no degradation occurs when ClpX is omitted
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UmuD/UmuD' heterodimer is more stable than each of the homodimers
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His-tag fusion protein, Ni-affinity chromatography
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homogeneity, wild-type and mutants
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Ni2+-NTA affinity resin column chromatography
Superdex 75 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21(DE3) cells
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expression in Escherichia coli
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fusion protein with beta-galactosidase
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gene umuD
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gene umuD, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)
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gene umuDAb, the umuD homologue umuDAb is present throughout the Acinetobacter genus and encodes an extra N-terminal region, transformation of Escherichia coli wild-type strain AB1157 and mutant cells with plasmids bearing various umuDAb alleles. UmuD is not required for UmuDAb expression from its native promoter, nor its disappearance after DNA damage through intermolecular interactions with Escherichia coli UmuD
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wild-type and mutant lacking the N-terminal extension
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
cellular level of UmuD increases approximately 10fold upon SOS induction
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the enzyme is expressed shortly after the induction of the SOS response
the mutant two complete umuDC operons (A1S_0636-A1S_0637 and A1S_1174-A1S_1173) and one unlinked umuD homolog (A1S_1389) determined in strain ATCC 17978 are induced by DNA damage and Rif
UmuD2 is up-regulated as part of the SOS response
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A83X
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the site-directed mutation of UmuDAb at Ala83 abolishes cleavage activity
K156X
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the site-directed mutation of UmuDAb at Lys156 abolishes cleavage activity
S119X
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the site-directed mutation of UmuDAb at Ser119 abolishes cleavage activity
A83X
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the site-directed mutation of UmuDAb at Ala83 abolishes cleavage activity
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K156X
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the site-directed mutation of UmuDAb at Lys156 abolishes cleavage activity
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S119X
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the site-directed mutation of UmuDAb at Ser119 abolishes cleavage activity
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A30T
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substantial extent of proteolytic cleavage
A89C
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reduced ability to for a heterodimer with UmuD'
C24Y
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poor extent of proteolytic cleavage
C25D
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site-directed mutagenesis, that removes the cleavage site Cys residue of UmuD, the mutation does not substantially affect UmuD function, cleavage site variant. For cleavage to occur, UmuD UmuD G25D dimer must first exchange in the presence of RecA:ssDNA, and any cleavage detected results from cleavage in trans. Cleavage is less efficient in this context, indicating that the decreased rate of cleavage in the trans dimers results from the time required for dimer exchange to first take place before cleavage can occur
D126C
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reduced ability to form a homodimer and a heterodimer with UmuD'
D20Y
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slight increase in activation rate by cleavage
D32C
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deficiencies in RecA-mediated cleavage as well as in UV mutagenesis, less than 30% of the wild-type activity
D3A
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the UmuD variant is non-cleavable but is a partial biological mimic of the cleaved form UmuD
D91A
the mutant is soluble and purifies as the wild type UmuD
D91K
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site-directed mutagenesis, the mutation abolishes the interaction between the enzyme and the DNA polymerase III alpha subunit
E11V/I12V/V13K
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supports ClpXP degradation of UmuD'
E35C
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deficiencies in RecA-mediated cleavage as well as in UV mutagenesis, less than 30% of the wild-type activity
F15A
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slight decrease of induced mutagenesis compared to wild-type
F15L
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no change in activation rate by cleavage
F18A
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decrease of induced mutagenesis to 20% of wild-type level, no cleavage of UmuD
F26A/P27A/S28A/P29A
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can form heterodimers and is recognized by ClpXP protease
G129D
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poor extent of proteolytic cleavage
G25D
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medium extent of proteolytic cleavage
G25S
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poor extent of proteolytic cleavage
G92C
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site-directed mutagenesis of of UmuD'
G92D
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substantial extent of proteolytic cleavage
G92K
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site-directed mutagenesis, the mutation abolishes the interaction between the enzyme and the DNA polymerase III alpha subunit
G92N
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defective for RecA-mediated UmuD cleavage
I38C
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poor reaction with iodoacetate
I4F
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slight increase in activation rate by cleavage
K97A
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mutant is able to undergo intermolecular cleavage, but not intramolecular self-cleavage
L101G/R102G
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mutant enzyme is defective in RecA-ssDNA-facilitated self-cleavage in vivo, can undergo RecA-ssDNA-facilitated cleavage in vitro, can interact directly with the RecA-ssDNA nucleoprotein filament in vitro, and is active in SOS mutagenesis in vivo
L107F
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substantial extent of proteolytic cleavage
L17F
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no change in activation rate by cleavage
L40C
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less than 30% of the wild-type activity, although defective in UV mutagenesis and in vitro RecA-mediated cleavage, mutant is able to be cleaved efficiently by RecA in vivo
L9A/R10A/E11A/I12A
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heterodimer with UmuD' displays a significant increase in stability
P48G
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expression of UmuD2 P48G is substantially lower than that of wild type UmuD2
Q23P
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mutant phenotype is reminiscent of the wild-type
Q23P/S60A
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UmuD is non-cleavable via an intramolecular cleavage pathway, but it remains cleavable via the intermolecular pathway
R37A
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when mutation is present in the UmuD' subunit of a UmuD/D' heterodimer it causes this subunit to be degraded substantially more slowly than its wild-type counterpart, when the mutation is present in the UmuD subunit of the heterodimer degradation of the UmuD' subunit occurs as efficiently as with the wild-type enzyme
R37C
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poor reaction with iodoacetate
S112C
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4-azidoiodoacetanilide-modified mutant, cross-links moderately efficiently with RecA
S19C
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4-azidoiodoacetanilide-modified mutant, almost no cross-linking with RecA
S81C
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4-azidoiodoacetanilide-modified mutant, cross-links most efficiently with RecA
T14A
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slight decrease of induced mutagenesis compared to wild-type
T14A/F15A/F18A
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decrease of induced mutagenesis to 20% of wild-type level, no cleavage of UmuD
T14A/L17A/F18A
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the mutant is a non-cleavable variant of UmuD
T14P
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no change in activation rate by cleavage
T95M
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substantial extent of proteolytic cleavage
V135S/K136A/R139A
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expression of UmuD2 V135S/K136A/R139A is substantially lower than that of wild type UmuD2
Y33C
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less than 30% of the wild-type activity, although defective in UV mutagenesis and in vitro RecA-mediated cleavage, mutant is able to be cleaved efficiently by RecA in vivo
C24A
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site-directed mutagenesis, that removes the cleavage site Cys residue of UmuD, the mutation does not substantially affect UmuD function, cleavage site variant
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C25D
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site-directed mutagenesis, that removes the cleavage site Cys residue of UmuD, the mutation does not substantially affect UmuD function, cleavage site variant. For cleavage to occur, UmuD UmuD G25D dimer must first exchange in the presence of RecA:ssDNA, and any cleavage detected results from cleavage in trans. Cleavage is less efficient in this context, indicating that the decreased rate of cleavage in the trans dimers results from the time required for dimer exchange to first take place before cleavage can occur
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G92C
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site-directed mutagenesis of of UmuD'
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N41D
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site-directed mutagenesis, the monomeric UmuD N41D variant can only cleave in the cis conformation
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S60A
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site-directed mutagenesis, a non-cleavable mutant of UmuD and UmuD', inactive active site mutant. For cleavage to occur, UmuD S60A dimer must first exchange in the presence of RecA:ssDNA, and any cleavage detected results from cleavage in trans. Cleavage is less efficient in this context, indicating that the decreased rate of cleavage in the trans dimers results from the time required for dimer exchange to first take place before cleavage can occur
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D91A
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the mutant is soluble and purifies as the wild type UmuD
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S60A
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noncleavable UmuD variant
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additional information