Information on EC 3.6.5.4 - signal-recognition-particle GTPase

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The expected taxonomic range for this enzyme is: Archaea, Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
3.6.5.4
-
RECOMMENDED NAME
GeneOntology No.
signal-recognition-particle GTPase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
GTP + H2O = GDP + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of phosphoric ester
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
NIL
-
-
SYSTEMATIC NAME
IUBMB Comments
GTP phosphohydrolase (protein-synthesis-assisting)
Activity is associated with the signal-recognition particle (a protein- and RNA-containing structure involved in endoplasmic-reticulum-associated protein synthesis).
CAS REGISTRY NUMBER
COMMENTARY hide
9059-32-9
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
strain 168
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-
Manually annotated by BRENDA team
strain 168
-
-
Manually annotated by BRENDA team
Chlamydomonas sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain MC1061
-
-
Manually annotated by BRENDA team
strain MRE600
-
-
Manually annotated by BRENDA team
WAM121
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain HPAG1
SwissProt
Manually annotated by BRENDA team
strain HPAG1
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
strain JDY819
-
-
Manually annotated by BRENDA team
strain HSC5
-
-
Manually annotated by BRENDA team
strain HSC5
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
-
in prokaryotic cells, the signal recognition particle consists of a SRP54 protein or Ffh and a 4.5S SRP RNA. Ffh contains a methionine-rich M domain, which binds the SRP RNA and the signal sequence on the translating ribosome. In addition, an NG domain in Ffh, comprising a GTPase G domain and a four-helix bundle N domain, forms a tight complex with a highly homologous NG domain in the SRP receptor, called FtsY in bacteria, in the presence of GTP. GTP hydrolysis at the end of the signal recognition particle cycle drives the disassembly of the Ffh-FtsY GTPase complex. The assembly of the signal recognition particle-FtsY GTPase complex and its GTPase activation require discrete conformational rearrangements in the signal recognition particle that are regulated by the RNC and the target membrane, respectively, thus ensuring the spatial and temporal precision of these molecular events during protein targeting, function of SRP RNA during co-translational protein targeting, overiew
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
GDP + H2O
GMP + phosphate
show the reaction diagram
GTP + H2O
GDP + phosphate
show the reaction diagram
guanylyl-5'-imidodiphosphate + H2O
?
show the reaction diagram
-
-
-
?
additional information
?
-
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
GDP + H2O
GMP + phosphate
show the reaction diagram
GTP + H2O
GDP + phosphate
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
LamB signal peptide
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dose-dependent inhibition of GTPase activity
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signal peptide
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isolated functional signal peptides bind nonspecifically to the RNA component of SRP and aggregate the entire signal recognition particle, leading to a loss of its intrinsic GTPase activity, this effect is an artifact of the high peptide concentrations and low salt conditions used in in vitro studies, signal sequences at the N-terminus of nascent chains in vivo do not exhibit this activity
-
additional information
-
membrane association of FtsY is stabilized by blocking its GTPase activity
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.5S RNA
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FtsY
-
signal recognition particle receptor
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SRP and its receptor stimulate each other’s GTPase activity, mechanism of reciprocal activation, substrate twinning activates the signal recognition particle and its receptor
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signal recognition particle RNA
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SRP RNA, the signal recognition particle RNA distal end triggers GTP hydrolysis in the signal recognition particle protein-SRP receptor GTPase, i.e. Ffh-FtsY GTPase, complex. An intact docking site at the distal end of SRP RNA is required to stimulate GTPase activation. Loop E plays a crucial role in GTPase activation by the SRP RNA
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.00081 - 0.0137
GTP
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.021 - 0.253
GTP
additional information
additional information
Escherichia coli
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kinetic data
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0002 - 0.00032
GDP
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.012
-
intrinsic GTPase activity
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7
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GTPase assay
8
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GTPase activity assay
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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GTPase activity assay
42
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GTPase assay
80
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GTPase assay
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4
subunit cpSRP43
5.93
-
calculated pI-value of the putative protein
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
-
the SRP targets the ribosome-nascent chain complexes to the inner membrane by interacting with the SRP receptor
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Escherichia coli (strain K12)
Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
31000
-
NG domain of Ffh, determined by SDS-PAGE
33760
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MALDI-MS
34900
apo-enzyme, analytical ultracentrifugation and small-angle X-ray scattering
35810
apo-enzyme, calculated from amino acid sequence
36000
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NG domain of FtsY, determined by SDS-PAGE
42200
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SRP receptor subunit alpha, DNA sequence analysis
47000
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MALDI-MS
50000
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gel filtration
50200
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calculated molecular mass of the putative protein
50400
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DNA sequence analysis
60000
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SDS-PAGE
68000
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SRP receptor subunit alpha
69000
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SRP receptor subunit alpha
400000
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FtsY-SecY translocon complex
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
subunit SRP54, X-ray crystallography
heterodimer
homodimer
monomer
additional information
-
homology modeling and structure analysis, overview
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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PAB0955 is able to phosphorylate itself in vitro at 80°C only in the presence of GTP and Mg2+ ions
ribonucleoprotein
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
subunit cpSRP43 in complex with a synthetic L18 peptide, hanging drop vapour diffusion method, using
the crystal structure of cpFtsY at 2.0 A resolution is reported
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the crystal structure of cpFtsY with bound malonate is solved at 1.75 A resolution
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signal recognition particle in complex with its receptor, X-ray diffraction structure determination and analysis at 3.94 A resolution
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hanging drop vapor diffusion method, 2.45 A crystal structure of the mammalian SRbeta in its Mg2+GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX
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crystal structure of the S-domain of signal-recognition-particle RNA at 2.6 A
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hanging drop vapor diffusion method, 2.45 A crystal structure of the mammalian SRbeta in its Mg2+GTP-bound state in complex with the minimal binding domain of SRalpha termed SRX
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ammonium sulfate precipitation or sodium citrate precipitation, structures of the NG domain of FtsY in two different forms: an apo and a sulfate-loaded form
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the crystal structure of PAB0955, free and in complex with six different nucleotides, is determined
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free and GDP-magnesium-bound forms, hanging drop vapour diffusion method, the hexagonal form grows in 1.1-1.5 M ammonium phosphate and 100 mM sodium acetate pH 5.0. The monoclinic form grows in 0.9-1.2 M lithium sulfate, 0.4-0.6 M ammonium sulfate, and 100 mM sodium citrate pH 5.0. For the GDP-bound structure, best crystals grow in 14-17% (w/v) PEG 8000 and 100 mM Tris pH 8.0
GDP-bound subunit SRP54 and free subunit SRP19 are crystallized by hanging drop vapour diffusion method, crystals of SRP54 grow in 1.0-1.3 M lithium sulfate and 100 mM sodium acetate pH 5.0, crystals of SRP19 grow in 1.2-1.3 M sodium malonate and 100 mM sodium acetate pH 5.0
hanging drop vapour diffusion method, at 21°C, using 50 mM cacodylic acid pH 6.5, 22% (w/v) PEG 4000, and 50 mM sodium acetate; hanging drop vapour diffusion method, at 21°C, using 50 mM cacodylic acid pH 6.5, 22% (w/v) PEG 4000, and 50 mM sodium acetate
crystal structure of SRP54 with and without its cognate RNA binding site
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crystal structures of the complex of signal recognition particle and signal recogition particle receptor show that the two GTPases associate via an unusually extensive and highly cooperative interaction surface and form a composite active site at the interface
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the 2.1 A X-ray structure of FtsY from Thermus aquaticus bound to GDP is reported
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the structure of the GMPPNP-stabilized complex of Thermus aquaticus Ffh and FtsY NG domains is determined at 1.97 A resolution
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two structures of the SRP GTPase Ffh NG-domains are determined at 1.1 A resolution providing the basis for comparative examination of the extensive water structure of the apo conformation
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X-ray structure of a complex of the N and G domains of Ffh with the GTPase FtsY of the SRP receptor in the presence of the non-hydrolyzable GTP analogue GMPPCP
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ffh NG is purified
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Ffh NG is purified, the Ffh NG-FtsY NGd20 complex is purified using ion exchange chromatography, a Q Sepharose and a SP Sepharose column; FtsY NGd20 is purified, the Ffh NG-FtsY NGd20 complex is purified using ion exchange chromatography, a Q Sepharose and a SP Sepharose column
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glutathione Sepharose column chromatography
heat selective precipitation, cobalt-chelating affinity chromatography, gel filtration, and ion-exchange chromatography
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HiPrep heparin Sepharose column chromatography, SP Sepharose column chromatography, and Superdex 75 gel filtration; HiPrep heparin Sepharose column chromatography, SP Sepharose column chromatography, and Superdex 75 gel filtration
Ni-Sepharose column chromatography
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proteins are purified using histidine-binding magnetic agarose beads
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recombinant wild-type and mutant cpSRP54 from Escherichia coli strain Rosetta BL21 by two steps of cation exchange chromatography
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the FtsY NGd20 protein is purified over a HiTrap Blue column, desalted, then passed over a HiTrap SP and a HiTrap Q Sepharose column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a construct of Thermus aquaticus FtsY in which the first 20 amino acids are deleted is subsequently expressed; the NG domain of Thermus aquaticus Ffh is cloned and subsequently expressed
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cpFtsY is cloned and expressed in Escherichia coli
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expressed in Escherichia coli
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expressed in Escherichia coli B834(DE3)-Rosetta2 cells
expressed in Escherichia coli BL21 (DE3) cells
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expressed in Escherichia coli BL21 cells
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expressed in Escherichia coli BL21(DE3) cells
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expressed in Escherichia coli BL21(DE3)-Rosetta2 cells
expressed in Escherichia coli BL21-AI cells; expressed in Escherichia coli BL21-AI cells
expressed in Escherichia coli BL21-CodonPlus(DE3) cells and in COS-1 cells
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expression in Escherichia coli
expression of wild-type and mutant cpSRP54 in Escherichia coli strain Rosetta BL21
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Ffh NG is expressed
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into the vectors pTD37 and pET22b
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N and G domains of Ffh, expression in Escherichia coli BL21(DE3)-Rosetta
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the Escherichia coli strain TOP10 is used for general cloning stategies and the strain SCS110 as an intermediary host in Bacillus cereus transformation experiments, the vectors pRN5101 and pDG148 are used
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the NG domain of Ffh is cloned into pUCm-T, and into pET-15b for expression in Escherichia coli BL21 cells; the NG domain of FtsY is cloned into pUCm-T, and into pET-15b for expression in Escherichia coli BL21 cells
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the vector pJGS3 expressing the protein FtsY NGd20, in which the N-terminal residues 2-20 of FtsY are deleted, is constructed by PCR-based site-directed mutagenesis of pTB88 for expression in Escherichia coli Rosetta-2DE3 pLysS cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D329A
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diminished GTPase activity
T326N
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diminished GTPase activity
Thr112Ala
A142W
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site-directed mutagenesis, the mutant cpSRP54 exhibits the same GTP-dependent complex assembly kinetics as wild-type cpSRP54
A143L
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
A143W
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
A168W
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
A169L
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
A169W
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
D137A
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site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
D163A
-
site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
F33L
-
mutation in the N-domain modulates the interaction kinetic between cpSRP54 and cpFtsY
F71V
-
mutation in the N-domain modulates the interaction kinetic between cpSRP54 and cpFtsY
L164Y
the mutant shows no interaction with synthetic L18 peptide
R140A
-
site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
R166A
-
site-directed mutagenesis, mutant cpSRP54 GTP-dependent complex assembly kinetics compared to the wild-type cpSRP54, overview
Y204A
the mutant shows no interaction with synthetic L18 peptide
DELTAflhF1
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flhF fragment from bp +282 to +1071 relative to the translational initiation site
DELTAflhF2
-
flhF fragment from bp +210 to +685 relative to the translational initiation site
D181N
-
hydrolysis of XTP favored over GTP
G118L
-
hydrolysis of XTP favored over GTP
G118L/D181N
-
hydrolysis of XTP favored over GTP
H119L
-
hydrolysis of XTP favored over GTP
K751/H119L
-
hydrolysis of XTP favored over GTP
K75I
-
hydrolysis of XTP favored over GTP
N178K
-
hydrolysis of XTP favored over GTP
A143W
-
mutant, GTPase activation defective
A144W
-
mutant, GTPase activation defective
A192D
-
reduced GTP hydrolysis, no effect on the interaction with FtsY
A254L
-
no growth defect
A334W
-
mutant, exhibits no significant GTP hydrolysis
A335W
-
mutant, GTPase activation defective
A336W
-
mutant, GTPase activation defective
C86A
-
mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, C86 and C86G reduce GTPase activity by only 50%
C86G
-
mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, DELTAC86 and C86G reduce GTPase activity by only 50%. Despite defective GTP hydrolysis, the G83A mutant shows any detectable defect in the efficiency of GTPase docking at the distal end
C86U
-
mutations at C86 yield a more complex pattern: whereas C86A and C86U completely abolish GTPase activation by the RNA, C86 and C86G reduce GTPase activity by only 50%
C87A/C97U
-
site-directed mutagenesis, combining C97U with C87A generates a superactive SRP RNA double mutant that hydrolyzes GTP 5.5fold faster than wild-type SRP RNA
C97U
-
site-directed mutagenesis, the mutant prolongs GTPase docking at the distal end, which correlates with its faster GTP hydrolysis rate
C97U/G99A
-
site-directed mutagenesis, combining G99A with C87A generate s a superactive SRP RNA double mutant that hydrolyzes GTP 4.6fold faster than wild-type SRP RNA
D253N
-
no growth defect
E475K
-
mutant, complex formation defective
F240V
-
mutation in the N-domain modulates the interaction kinetic between Ffh and FtsY
G110S
-
reduced GTP hydrolysis, no effect on the interaction with FtsY
G256A
-
no growth defect
G257A
-
residue resides at the N-GTPase domain interface, mutation produces a lethal phenotype, it does not significantly affect Ffh function, but severely reduces interaction with FtsY
G455V
-
mutant, defective in SRP-FtsY complex formation
G83A
-
deletion or substitution of G83 by any other nucleotide completely abolishes the stimulatory effect of the SRP RNA on GTP hydrolysis. Despite defective GTP hydrolysis, the G83A mutant shows any detectable defect in the efficiency of GTPase docking at the distal end
G99A
-
site-directed mutagenesis, the mutant prolongs GTPase docking at the distal end, which correlates with its faster GTP hydrolysis rate
K399A
-
mutant, complex formation defective
L195P
-
reduced GTP hydrolysis, no effect on the interaction with FtsY
L199F
-
mutation in the N-domain modulates the interaction kinetic between Ffh and FtsY
N302A
-
mutant, GTPase activation defective
P142L
-
reduced GTP hydrolysis, no effect on the interaction with FtsY
Q109A
-
mutant, GTPase activation defective
R141A
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mutant, GTPase activation defective
R194A
-
mutant, GTPase activation defective
R255N
-
no growth defect
R333A
-
mutant, GTPase activation defective
R386A
-
mutant, GTPase activation defective
T307A
-
mutant, complex formation and GTPase activation defective
A254L
-
no growth defect
-
D253N
-
no growth defect
-
G256A
-
no growth defect
-
G257A
-
residue resides at the N-GTPase domain interface, mutation produces a lethal phenotype, it does not significantly affect Ffh function, but severely reduces interaction with FtsY
-
R255N
-
no growth defect
-
F48A
-
the cpFtsY mutant exhibits a GTP hydrolysis rate 4times greater than wild type cpFtsY in the absence of liposomes, the mutation reduces light-harvesting chlorophyll-binding protein integration efficiency by nearly 80%
F49A
-
the mutation reduces light-harvesting chlorophyll-binding protein integration efficiency by nearly 40%
E157Q
-
reduced affinity for GTP
H91L
-
reduced GTPase activity
H91L/E157Q
-
mutant
H91L/N154A/E157A
-
mutant
H91L/N154I
-
mutant
K51A/T52A
-
mutant
K51A/T52A/E157Q
-
mutant
K51A/T52A/H91L
-
mutant
K51I
-
reduced nucleotide affinity
K51I/H91L
-
mutant
K51I/N154I
-
mutant
N154A/E157A
-
mutant
N154I
-
impaired nucleotide exchange
P46A/Q47A/N48A/DELTAS49
-
mutant
S220A
-
bypass requirement for GEF
S49A
-
reduced GTP hydrolysis
T52N
-
increased affinity for GEF, reduced affinity for GTP
T66A
-
prevents GTP-dependent interaction with GAP
A145S
-
substitution introduced into the putative GTP binding motif GXXGXGK
G141V
-
substitution introduced into the putative GTP binding motif GXXGXGK
G222V
-
substitution introduced into the putative GTP binding motif GXXGXGK
GAK-VSG
-
substitution introduced into the putative GTP binding motif GXXGXGK
GTK-VSG
-
substitution introduced into the putative GTP binding motif GXXGXGK
K147G
-
substitution introduced into the putative GTP binding motif GXXGXGK
K228G
-
substitution introduced into the putative GTP binding motif GXXGXGK
T226S
-
substitution introduced into the putative GTP binding motif GXXGXGK
additional information
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
mutant cpFtsY is refolded after treatment with 8 M urea
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine