Information on EC 3.6.3.B1 - bacterial protein-transporting ATPase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.6.3.B1
preliminary BBRENDA supplied EC number
RECOMMENDED NAME
GeneOntology No.
bacterial protein-transporting ATPase
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
transmembrane transport
SYSTEMATIC NAME
IUBMB Comments
ATP phosphohydrolase (bacterial protein transporting)
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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UniProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
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
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + Msmeg1704 in
ADP + phosphate + Msmeg1704 out
show the reaction diagram
ATP + H2O + Msmeg1704/in
ADP + phosphate + Msmeg1704/out
show the reaction diagram
-
-
-
ir
ATP + H2O + Msmeg1712/in
ADP + phosphate + Msmeg1712/out
show the reaction diagram
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-
-
ir
ATP + H2O + pOA/in
ADP + phosphate + pOA/out
show the reaction diagram
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pOA, a truncated form of proOmpA with a new cysteine to allow for disulfide-loop formation
the disulfide-loop renders pOA unable to pass the channel, adding of DTT completes the translocation
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ir
ATP + H2O + proOmpA/in
ADP + phosphate + proOmpA/out
show the reaction diagram
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-
-
-
ir
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
ATP + H2O
ADP + phosphate
show the reaction diagram
ATP + H2O + Msmeg1704 in
ADP + phosphate + Msmeg1704 out
show the reaction diagram
ATP + H2O + Msmeg1704/in
ADP + phosphate + Msmeg1704/out
show the reaction diagram
A0QYG9
-
-
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ir
ATP + H2O + Msmeg1712/in
ADP + phosphate + Msmeg1712/out
show the reaction diagram
A0QYG9
-
-
-
ir
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Mg2+
ATP hydrolysis activity of the recombinant protein 6His-SecA2 aquires at least 0.5 mM Mg2+ and is higher with 2.5 mM Mg2+; ATP hydrolysis activity of the recombinant protein 6His-SecA is highest with 0.1 mM Mg2+ and decreases both with higher and lower concentration of Mg2+
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,2'-(alpha,alpha'-xylene)bis(sulfanediyl)bis-(6-(4-bromophenyl)-5-cyano-4-oxopyrimidine)
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2,2'-(alpha,alpha'-xylene)bis(sulfanediyl)bis-(6-(biphenyl-4-yl)-5-cyano-4-oxopyrimidine)
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compound does not show antimicrobial activity
2-((4-azidobenzyl)thio)-4-(4-(benzyloxy)phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
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good antimicrobial inhibition with MIC of 12.5 microM
2-((4-azidobenzyl)thio)-6-oxo-4-(4-phenoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile
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good antimicrobial inhibition with MIC of 18.2 microM
2-(benzylsulfanyl)-4-(biphenyl-4-yl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
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exhibits most potent inhibition effects against strain NR698 with increased outer membrane permeability
azide
1.5 mM azide decrease the transport of the cell-surface glycoprotein mutant GspB736flag, 15 mM nearly prevent transport of GspB736flag, azide have no effect on ATP hydrolysis activity of the recombinant protein 6His-SecA; 1.5 mM azide markedly decrease the transport of the cell-surface glycoprotein mutant GspB736flag, 15 mM nearly prevent transport of GspB736flag, azide have no effect on ATP hydrolysis activity of the recombinant protein 6His-SecA2
eosin Y
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erythrosin B
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the potency in inhibiting the truncated SecA ATPase correlates with the ability to inhibit the biologically relevant protein translocation activity of SecA and also translates into antibacterial effects
Rose bengal
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the potency in inhibiting the truncated SecA ATPase correlates with the ability to inhibit the biologically relevant protein translocation activity of SecA and also translates into antibacterial effects
additional information
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Insertion of charged amino acid residues into the preprotein proOmpA strongly inhibits SecA translocation ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues.
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Calcium
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27-82 micromol Ca2+ enhances ATPase activity in presence of Escherichia coli PE/anionic phospholipid membranes up to 3-fold
DTT
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effect on mutant N95CC, a large stimulation of ATPase activity is only observed when both proOmpA and DTT are present
Phospholipid
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stimulated up to 4fold by phospholipids with an optimum at 74°C. Membrane vesicles and proteoliposomes containing SecYE and SecYEG support 2fold to 4fold stimulation of the precursor dependent SecA ATPase activity
proOmpA
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effect on mutant N95CC, a large stimulation of ATPase activity is only observed when both proOmpA and DTT are present
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SecM
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SecA synthesized without the signal sequence on SecM is much less active on average than that synthesized in combination with normal SecM. The elongation arrest per se is not sufficient in terms of the SecA-activating ability of SecM. Targeting of nascent SecM to the membrane translocon is another crucial feature for SecM to enhance SEcA activity
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.05 - 0.263
ATP
additional information
additional information
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.002 - 0.02
2,2'-(alpha,alpha'-xylene)bis(sulfanediyl)bis-(6-(4-bromophenyl)-5-cyano-4-oxopyrimidine)
0.002 - 0.05
2,2'-(alpha,alpha'-xylene)bis(sulfanediyl)bis-(6-(biphenyl-4-yl)-5-cyano-4-oxopyrimidine)
0.0024
2-((4-azidobenzyl)thio)-4-(4-(benzyloxy)phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
Escherichia coli
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37°C, pH not specified in the publication
0.0022
2-((4-azidobenzyl)thio)-6-oxo-4-(4-phenoxyphenyl)-1,6-dihydropyrimidine-5-carbonitrile
Escherichia coli
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37°C, pH not specified in the publication
0.06
2-(benzylsulfanyl)-4-(biphenyl-4-yl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
Escherichia coli
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recombinant SecA, pH 7.6, 40°C
0.025
eosin Y
Escherichia coli
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truncated SecA without the C-terminal regulatory domain, 40°C, pH 7.6
0.002
erythrosin B
Escherichia coli
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truncated SecA without the C-terminal regulatory domain, 40°C, pH 7.6
0.0005
Rose bengal
Escherichia coli
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truncated SecA without the C-terminal regulatory domain, pH 7.6, 40°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
PaSecAN236, liposome
0.0065
PaSecAL43P, liposome; PaSecAN236, PaSecAL43P
0.052
PaSecAN236, PaSecAL43P, liposome
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
ATPase activity assay
7.6
ATPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
ATPase activity assay
37
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ATPase and translocation assay
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
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SecA-dependent secretion and SrtA anchoring machineries are spatially coupled and localized at the division septum in exponentially growing cultures
Manually annotated by BRENDA team
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SecY channel
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Manually annotated by BRENDA team
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inner membrane vesicle
Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27000
mutant PaSecAN236, determined by SDS-PAGE
54000
Streptococcus parasanguis strain VT1697, expressed from plasmid, deletion mutant, residues 1-330 are deleted
64000
Streptococcus parasanguis strain VT1574, expressed from chromosomal locus, deletion mutant, residues 331-583 are deleted, 83.6% in the membrane; Streptococcus parasanguis strain VT1584, expressed from chromosomal locus, deletion mutant
65000
Streptococcus parasanguis strain VT1694, expressed from plasmid, deletion mutant, residues 578-797 are deleted, 76.6% in the membrane
79000
Streptococcus parasanguis strain VT1584, expressed from plasmid, deletion mutant, residues 678-779 are deleted, 68.0% in the membrane
81000
Streptococcus parasanguis strain VT1583, expressed from chromosomal locus, deletion mutant, residues 590-676 are deleted; Streptococcus parasanguis strain VT1700, expressed from chromosomal locus, deletion mutant, residues 590-676 are deleted; Streptococcus parasanguis strain VT1702, expressed from chromosomal locus, deletion mutant, residues 590-676 are deleted
82000
Streptococcus parasanguis strain VT1702, expressed from plasmid, deletion muatnt, 1-330 are deleted, GFP-tag, 84.4% in the membrane
85400
monomer, predicted molecular mass, determined by SDS-PAGE
92000
Streptococcus parasanguis strain FW213, expressed from chromosomal locus, full-length protein, 95.5% in the membrane; Streptococcus parasanguis strain VT1694, expressed from chromosomal locus, full-length protein; Streptococcus parasanguis strain VT1697, expressed from chromosomal locus, full-length protein
93000
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mutant SecAdelta11/N95, determined by SDS-PAGE
95000
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determined by SDS-PAGE
101000
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monomer, predicted molecular mass
102000
106000
monomer, predicted molecular mass, determined by SDS-PAGE
109000
Streptococcus parasanguis strain VT1700, expressed from plasmid, deletion mutant, residues 590-676 are deleted, GFP-tag, 86.8% in the membrane
200000
204000
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dimer, gel filtration coupled with static light scatter
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals diffracting to 2.4 A resolution are obtained using the vapour-diffusion technique with sitting drops. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 A, alpha = gamma = 90.0°, beta = 119°. A selenomethionine derivative is prepared
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molecular docking of inhibitor 2-((4-azidobenzyl)thio)-4-(4-(benzyloxy)phenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile. The azido group points towards hydrophilic residues including A/Asp512, A/Arg509, A/Gly510 and A/Thr511. The torsion around the -CH2O- atoms between the two phenyl groups allows the terminal phenyl group to rest in a pocket away from the hydrophilic residues B/Thr511, B/Gly510 and B/Glu487. Most of the active inhibitors seem to bind at the interface of chains A and B
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to 1.9 A resolution. Structure represents an intermediate state during the transition of the clamp from an open to a closed conformation. Closure of the clamp occurs in two phases, an initial movement of polypeptide cross-linking domain PPXD, helical scaffold domain HSD, and helical wing domain HWD as a unit, followed by a movement of PPXD alone toward nucleotide-binding domain NBD2. The substrate associates with the back of the clamp by dynamic hydrogen bonding and the clamp is laterally closed by a conserved loop of the PPXD
2.8 A resolution crystal structure
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crystallization by hanging-drop vapour-diffusion technique in two different space groups P2(1(2))21 (a = b = 168.6 A, c = 149.8 A) and P6(1(5))22 (a = b = 130.9 A, c = 564.6 A). The crystals, improved by macroseeding, diffract to beyond 2.8 A and 3.5 A resolution for the trigonal and hexagonal crystal forms, respectively
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; using a His Bind purification kit
inverted membrane vesicles are prepared
using a Blue-Sepharose CL-6B column; using a Blue-Sepharose CL-6B column
using a Q-Sepharose 26/10 anion-exchange column, a SP-Sepharose 16/5 cation exchange column, and a Superose 12 HR gel filtration column
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using osmotic shock, a SP-Sepharose 26/10 packed cation-exchange column, and a Sephacryl-200 HR 16/90 packed column
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by SecA-overproducing Escherichia coli strain RR1/pMAN400
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different plasmids are used, Ara and Lac promoter controlled
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expressed in a SecA-overproducing Escherichia coli strain
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expressed in Escherichia coli after stimulation with 1 mM isopropyl-beta-D-thiogalactoside
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expressed in Escherichia coli harboring a special plasmid
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expressed in Escherichia coli strain BL21(DE3) harboring a special plasmid
expressed in Escherichia coli strain Bl21DE3 as a recombinant C-terminal His6-tagged protein
expressed in Escherichia coli using special plasmids
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expression in Escherichia coli; expression in Escherichia coli
expression of a truncated form of SecA lacking the C-terminal inhibitory domain
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into the pET-27b+ vector for expression in Escherichia coli BL21DE3 cells; into the pET-27b+ vector for expression in Escherichia coli BL21DE3 cells
into the pET29a vector for expression in Escherichia coli BL21DE3 cells; into the pET41b vector for expression in Escherichia coli BL21DE3 cells
into the pET5a vector for expression in Escherichia coli BL21DE3 cells
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overexpression in Escherichia coli
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overexpression in Escherichia coli strain XL-1 Blue
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plasmid pT7secA-Cys-0, a derivative of pT7secA2 that has all four cysteine codons within secA changed to serine, is used to create the monocysteine secA mutants
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subcloned in Escherichia coli; subcloned in Escherichia coli
the DNA fragment encoding the N-terminal 236 amino acid residues is cloned into the pET20b vector for expression in Escherichia coli BL21.19 cells
the secA2 gene of Mycobacterium smegmatis is cloned from pMB208 into pMV306.kan to generate pYA810
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D209N
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mutant of SecA capable of binding, but not hydrolysing ATP
E210Q
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mutant of SecA capable of binding, but not hydrolysing ATP
Cys-less
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mutant for analyzing the interaction and regulatory domains of SecA
D209A
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a dominant-negative mutant, binds ATP but is unable to hydrolyze it and is inactive in proOmpA translocation. Mutant generates a translocation intermediate of 18 kDa. Further addition of wild-type SecA causes its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation causes a topology inversion of SecG
E400C
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mutant for analyzing the interaction and regulatory domains of SecA
E400C/R642C
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mutant for analyzing the interaction and regulatory domains of SecA
E400R
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/A628T
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/E619K
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/H620P
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/I627T
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/L610P
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/M607T
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mutant for analyzing the interaction and regulatory domains of SecA
E400R/N629D
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mutant for analyzing the interaction and regulatory domains of SecA
E635C
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mutant for analyzing the interaction and regulatory domains of SecA
Ile3A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
K108R
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mutant, defective in ATP binding and protein translocation in vitro, as well as biologically inactive in vivo
L2A/I3A
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mutation does not substantially affect SecA dimerization
Leu2A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
Leu5A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
Leu6A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
N95
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truncated version of Escherichia coli SecA, the last 70 residues are lacking
N95CC
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two cysteines are introduced into a truncated version of Escherichia coli SecA, at position 636 and 801, the last 70 residues are lacking, mutant is dimeric and fully functional
Phe10A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
R400R/M607T
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mutant for analyzing the interaction and regulatory domains of SecA
R642C
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mutant for analyzing the interaction and regulatory domains of SecA
R642E
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/A628T
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/E619K
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/H620P
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/I627T
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/L610P
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/M607T
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mutant for analyzing the interaction and regulatory domains of SecA
R642E/N629D
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mutant for analyzing the interaction and regulatory domains of SecA
R656C
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mutant for analyzing the interaction and regulatory domains of SecA
SecAA
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tandem SecA
SecADELTA11/N95
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monomeric SecA derivative mutant, which lacks extreme terminal residues and shows in vitro and in vivo activities
V9A/F10A
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mutation enhances dissociation by 8fold with respect to that of wild-type SecA
Val9A
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mutation completely blocks dimerization of SecA in 300 mM KCl buffer
K115A
mutant, substitution of the conserved lysine in the Walker A motif eliminates ATP binding and affects the biological activity
K115R
mutant, substitution of the conserved lysine in the Walker A motif eliminates ATP binding and affects the biological activity
K115A
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mutant, substitution of the conserved lysine in the Walker A motif eliminates ATP binding and affects the biological activity
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K115R
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mutant, substitution of the conserved lysine in the Walker A motif eliminates ATP binding and affects the biological activity
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PaSecAN236
N-terminal 236 amino acids of PaSecA
SecA2DELTA1-990
mutant, cloned into different plamids, construction of different strains
SecA2DELTA1732-2397 7
mutant, cloned into different plamids, construction of different strains
SecA2DELTA1768-2028
mutant, cloned into different plamids, construction of different strains
SecA2DELTA991-1749
mutant, cloned into different plamids, construction of different strains
E485R/E488R/R367E
mutation leads to a closed conformation of the clamp, the C-loop remains inside the clamp
R367E
mutation of a conserved residue, results in significant lateral opening of the clamp, which leads to increased dissociation of the substrate
E485R/E488R/R367E
-
mutation leads to a closed conformation of the clamp, the C-loop remains inside the clamp
-
R367E
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mutation of a conserved residue, results in significant lateral opening of the clamp, which leads to increased dissociation of the substrate
-
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
SecA2-His6 protein is purified from inclusion bodies and refolded