Information on EC 2.4.2.36 - NAD+-diphthamide ADP-ribosyltransferase

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

EC NUMBER
COMMENTARY hide
2.4.2.36
-
RECOMMENDED NAME
GeneOntology No.
NAD+-diphthamide ADP-ribosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
NAD+ + diphthamide-[translation elongation factor 2] = nicotinamide + N-(ADP-D-ribosyl)diphthamide-[translation elongation factor 2]
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pentosyl group transfer
SYSTEMATIC NAME
IUBMB Comments
NAD+:diphthamide-[translation elongation factor 2] N-(ADP-D-ribosyl)transferase
Diphtheria toxin and some other bacterial toxins catalyse this reaction, which inactivates translation elongation factor 2 (EF2). The acceptor is diphthamide, a unique modification of a histidine residue in the elongation factor found in archaebacteria and all eukaryotes, but not in eubacteria. cf. EC 2.4.2.31 NAD(P)+---protein-arginine ADP-ribosyltransferase. The relevant histidine of EF2 is His715 in mammals, His699 in yeast and His600 in Pyrococcus horikoshii.
CAS REGISTRY NUMBER
COMMENTARY hide
52933-21-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
baby
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-
Manually annotated by BRENDA team
strain 569B, hyper-producer of cholera toxin
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-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
diethylamino(benzylidine-amino)guanidine + NAD+
?
show the reaction diagram
-
DEABAG
-
-
?
eEF2 + NAD+
eEF2 N-(ADP-D-ribosyl)diphthamide + NADH + H+
show the reaction diagram
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
show the reaction diagram
NAD+ + G-protein
?
show the reaction diagram
-
NAD-dependent ADP-ribosylation of Arg201 of G-protein alpha-subunit, this locks the G-protein in its active state producing cAMP
-
-
?
NAD+ + peptide diphthamide
nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide
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
eEF2 + NAD+
eEF2 N-(ADP-D-ribosyl)diphthamide + NADH + H+
show the reaction diagram
-
i.e. eukaryotic elongation factor 2, contains a post-translationally modified histidine residue, known as diphthamide, which is the specific ADP-ribosylation target of Pseudomonas aeruginosa exotoxin A
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-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
show the reaction diagram
P11439
transfer of ADP-ribose moiety (oxocarbenium ion) of NAD+ onto N3 of diphthamide imidazole of essential ribosomal elongation factor 2 (eEF2)
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-
?
NAD+ + G-protein
?
show the reaction diagram
-
NAD-dependent ADP-ribosylation of Arg201 of G-protein alpha-subunit, this locks the G-protein in its active state producing cAMP
-
-
?
NAD+ + peptide diphthamide
nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide
show the reaction diagram
-
-
-
-
?
additional information
?
-
P11439
NAD+-dependent ADP-ribosyltransfer (ADPRT) including NAD+-glycohydrolysis (GH) activity, deadly virulent due to covalent modification and thus inactivation of proteins that are essential for the host
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-
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,8-naphthalimide
-
potent competitive
3-((3,3-diethylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 24% inhibition
3-((3,3-diisopropylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 94% inhibition
3-((3-(1-benzylpiperidin-4-yl)guanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 96% inhibition
3-((3-(1-benzylpyrrolidin-3-yl)guanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 68% inhibition
3-((3-(2-(ethyl(m-tolyl)amino)ethyl)guanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 76% inhibition
3-((3-(2-morpholinoethyl)guanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 57% inhibition
3-((3-(4-bromobenzyl)guanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 98% inhibition
3-((3-(4-methoxybenzyl)guanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 97% inhibition, not sufficient material for IC50 and Ki estimation
3-((3-benzylguanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 93% inhibition
3-((3-phenethylguanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 93% inhibition
3-((3-phenylguanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 57% inhibition
3-((3-tert-butylguanidino)methyl)benzamide
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1 mM, 37°C, pH 7, 39% inhibition
3-(3,3-diisopropylguanidino)benzamide
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1 mM, 37°C, pH 7, 15% inhibition
3-(3-(1-benzylpiperidin-4-yl)guanidino)benzamide
-
1 mM, 37°C, pH 7, 58% inhibition
3-(3-(4-bromobenzyl)guanidino)benzamide
-
1 mM, 37°C, pH 7, 22% inhibition
3-(3-benzylguanidino)benzamide
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1 mM, 37°C, pH 7, 10% inhibition
3-(3-phenylguanidino)benzamide
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1 mM, 37°C, pH 7, 12% inhibition
3-(3-tert-butylguanidino)benzamide
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1 mM, 37°C, pH 7, 77% inhibition
4-amino-1,8-naphthalimide
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Cytoplasmic extract of pyBHK-cells
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not fragment A
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Elastase
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inactivation follows pseudo-first order kinetic
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ethyl 4-(3-(3-carbamoylbenzyl)guanidino)piperidine-1-carboxylate
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1 mM, 37°C, pH 7, 34% inhibition
histamine
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250 mM, almost complete inhibition
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
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activation, together with SDS or guanidine hydrochloride
cysteine
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activation, together with SDS or guanidine hydrochloride
dithiothreitol
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enhances activity together with urea
guanidine hydrochloride
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enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite
SDS
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enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite
sulfite
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activation, together with SDS or guanidine hydrochloride
Urea
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enhances activity together with dithiothreitol
additional information
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exotoxin is synthesized in a catalytically inactive, proenzyme form, unfolding of the toxin molecule may cause activation
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4
diethylamino(benzylidine-amino)guanidine
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-
0.02
elongation factor 2
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-
0.275 - 14
NAD+
additional information
NAD+
double mutant E546A/R551A, relative KD = 1.11 +/-0.03; double mutant E546D/R551K, relative KD = 2.71 +/-0.74; double mutant E546R/R551E, relative KD = 1.29 +/-0.49; mutant D461A, KD = 1.03 +/-0.02 relative to wild-type (c); mutant D463A, KD = 1.18 +/-0.04 relative to wild-type (c); mutant E546A, relative KD = 1.77 +/-0.34; mutant E546D, relative KD = 3.83 +/-0.89; mutant E546F, relative KD = 1.97 +/-0.11; mutant E546H, relative KD = 1.03 +/-0.09; mutant E546N, relative KD = 2.14 +/-0.26; mutant E546Q, relative KD = 2.31 +/-0.71; mutant E547A, relative KD = 1.97 +/-0.77; mutant E548A, relative KD = 2.69 +/-0.11; mutant E553A, relative KD = 2.57 +/-0.86; mutant G453A, KD = 4.85 +/-1.07 relative to wild-type (c); mutant G454A, KD = 6.25 +/-0.22 relative to wild-type (c); mutant G549A, relative KD = 8.00 +/-0.94; mutant G550A, relative KD = 6.60 +/-0.54; mutant L462A, KD = 1.86 +/-0.10 relative to wild-type (c); mutant L552A, relative KD = 4.43 +/-0.57; mutant Q460A, KD = 4.30 +/-0.43 relative to wild-type (c); mutant R456A, KD = 3.56 +/-0.53 relative to wild-type (c); mutant R458A, KD = 5.78 +/-0.18 relative to wild-type (c); mutant R458H, KD = 31.02 +/-7.18 relative to wild-type (d); mutant R458K, KD = 3.18 +/-0.23 relative to wild-type (c); mutant R458Q, KD = 0.45 +/-0.06 relative to wild-type (e); mutant R458W, KD = 11.26 +/-1.16 relative to wild-type (d); mutant R551A, relative KD = 2.69 +/-0.03; mutant R551C, relative KD = 5.57 +/-0.91; mutant R551E, relative KD = 5.03 +/-0.51; mutant R551H, relative KD = 2.29 +/-0.46; mutant R551K, relative KD = 1.29 +/-0.03; mutant R551Q, relative KD = 2.63 +/-0.39; mutant S459A, KD = 1.59 +/-0.16 relative to wild-type (c); mutant V455A, KD = 3.31 +/-0.24 relative to wild-type (c); wild-type, KD = 35 +/-3 microM, relative KD = 1.00 +/-0.09, estimated according to quenched intrinsic tryptophan fluorescence upon NAD+ binding to the active site; wild-type, KD = 36 +/-8 microM (c), KD = 62 +/-2 microM (d), KD = 74 +/-16 microM, relative KD = 1.00 +/-0.03
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
12
elongation factor 2
Pseudomonas aeruginosa
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11
NAD+
Pseudomonas aeruginosa
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-
additional information
NAD+
Pseudomonas aeruginosa
P11439
0.0062-0.13/min, wild-type GH activity, in the absence of eEF2, pH 7.9, 25C, 60 min; 0.125 +/-0.007/min (a), wild-type GH activity, in the absence of eEF2, pH 7.9, 25C, 60 min; 0.130 +/-0.012/min (b), wild-type GH activity, in the absence of eEF2, pH 7.9, 25C, 60 min; 1306 +/-121/min (a), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; 1495 +/-224/min (b), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; 628 +/-8/min (B), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; 746 +/-18/min (A), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; 847 +/-21/min (C), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; 900 +/-20/min (D), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25C, 300 sec; double mutant E546A/R551A, kcat = 0.0001 +/-0.0001, relative to wild-type ADPRT activity (C); double mutant E546A/R551A, kcat = 0.84 +/-0.04, relative to wild-type GH activity; double mutant E546D/R551K, kcat = 0.0009 +/-0.0013, relative to wild-type ADPRT activity (C); double mutant E546D/R551K, kcat = 0.42 +/-0.0002, relative to wild-type GH activity; double mutant E546R/R551E, kcat = 0.00001 +/-0.000002, relative to wild-type ADPRT activity (C); double mutant E546R/R551E, kcat = 0.38 +/-0.01, relative to wild-type GH activity; mutant D461A, kcat = 1.001 +/-0.091, relative to wild-type ADPRT activity (a); mutant D461A, kcat = 1.076 +/-0.041, relative to wild-type GH activity (a); mutant D463A, kcat = 1.148 +/-0.038, relative to wild-type GH activity (a); mutant D463A, kcat = 1.270 +/-0.204, relative to wild-type ADPRT activity (a); mutant E546A, kcat = 0.0012 +/-0.00002, relative to wild-type ADPRT activity (A); mutant E546A, kcat = 0.24 +/-0.008, relative to wild-type GH activity; mutant E546D, kcat = 0.00025 +/-0.00005, relative to wild-type ADPRT activity (B); mutant E546D, kcat = 0.45 +/-0.002, relative to wild-type GH activity; mutant E546F, kcat = 0.007 +/-0.0002, relative to wild-type ADPRT activity (D); mutant E546F, kcat = 0.69 +/-0.05, relative to wild-type GH activity; mutant E546H, kcat = 0.0012 +/-0.0002, relative to wild-type ADPRT activity (B); mutant E546H, kcat = 0.59 +/-0.009, relative to wild-type GH activity; mutant E546N, kcat = 0.0010 +/-0.00009, relative to wild-type ADPRT activity (B); mutant E546N, kcat = 0.67 +/-0.03, relative to wild-type GH activity; mutant E546Q, kcat = 0.011 +/-0.002, relative to wild-type ADPRT activity (B); mutant E546Q, kcat = 0.80 +/-0.04, relative to wild-type GH activity; mutant E547A, kcat = 0.795 +/-0.077, relative to wild-type ADPRT activity (A); mutant E547A, kcat = 0.83 +/-0.14, relative to wild-type GH activity; mutant E548A, kcat = 0.76 +/-0.01, relative to wild-type GH activity; mutant E548A, kcat = 1.669 +/-0.298, relative to wild-type ADPRT activity (A); mutant E553A, kcat = 0.0015 +/-0.00005, relative to wild-type ADPRT activity (A); mutant E553A, kcat = 0.07 +/-0.009, relative to wild-type GH activity; mutant G453A, kcat = 0.290 +/-0.040, relative to wild-type ADPRT activity (a); mutant G453A, kcat = 0.331 +/-0.019, relative to wild-type GH activity (a); mutant G454A, kcat = 0.031 +/-0.003, relative to wild-type GH activity (a); mutant G454A, kcat = 0.046 +/-0.008, relative to wild-type ADPRT activity (a); mutant G549A, kcat = 0.30 +/-0.005, relative to wild-type GH activity; mutant G549A, kcat = 0.770 +/-0.076, relative to wild-type ADPRT activity (A); mutant G550A, kcat = 0.41 +/-0.04, relative to wild-type GH activity; mutant G550A, kcat = 0.562 +/-0.166, relative to wild-type ADPRT activity (A); mutant L462A, kcat = 0.268 +/-0.041, relative to wild-type ADPRT activity (a); mutant L462A, kcat = 0.955 +/-0.015, relative to wild-type GH activity (a); mutant L552A, kcat = 0.58 +/-0.002, relative to wild-type GH activity; mutant L552A, kcat = 2.365 +/-0.527, relative to wild-type ADPRT activity (A); mutant Q460A, kcat = 0.115 +/-0.010, relative to wild-type ADPRT activity (a); mutant Q460A, kcat = 0.622 +/-0.014, relative to wild-type GH activity (a); mutant R456A, kcat = 0.230 +/-0.010, relative to wild-type GH activity (a); mutant R456A, kcat = 0.239 +/-0.034, relative to wild-type ADPRT activity (a); mutant R458A, kcat = 0.132 +/-0.031, relative to wild-type ADPRT activity (a); mutant R458A, kcat = 0.249 +/-0.021, relative to wild-type GH activity (a); mutant R458H, kcat = 0.018 +/-0.003, relative to wild-type GH activity (b); mutant R458H, kcat = 0.019 +/-0.007, relative to wild-type ADPRT activity (b); mutant R458K, kcat = 0.428 +/-0.033, relative to wild-type GH activity (a); mutant R458K, kcat = 0.543 +/-0.155, relative to wild-type ADPRT activity (a); mutant R458Q, kcat = 0.650 +/-0.130, relative to wild-type ADPRT activity (b); mutant R458Q, kcat = 0.991 +/-0.017, relative to wild-type GH activity (b); mutant R458W, kcat = 0.379 +/-0.043, relative to wild-type ADPRT activity (b); mutant R458W, kcat = 0.438 +/-0.040, relative to wild-type GH activity (b); mutant R551A, kcat = 0.32 +/-0.005, relative to wild-type GH activity; mutant R551A, kcat = 0.380 +/-0.024, relative to wild-type ADPRT activity (A); mutant R551C, kcat = 0.10 +/-0.006, relative to wild-type GH activity; mutant R551C, kcat = ~0, relative to wild-type ADPRT activity (B); mutant R551E, kcat = 0.011 +/-0.005, relative to wild-type ADPRT activity (B); mutant R551E, kcat = 0.09 +/-0.001, relative to wild-type GH activity; mutant R551H, kcat = 0.41 +/-0.002, relative to wild-type GH activity; mutant R551H, kcat = ~0, relative to wild-type ADPRT activity (B); mutant R551K, kcat = 0.317 +/-0.024, relative to wild-type ADPRT activity (B); mutant R551K, kcat = 0.69 +/-0.03, relative to wild-type GH activity; mutant R551Q, kcat = 0.185 +/-0.014, relative to wild-type ADPRT activity (B); mutant R551Q, kcat = 0.38 +/-0.01, relative to wild-type GH activity; mutant S459A, kcat = 1.119 +/-0.054, relative to wild-type GH activity (a); mutant S459A, kcat = 1.256 +/-0.144, relative to wild-type ADPRT activity (a); mutant V455A, kcat = 0.241 +/-0.032, relative to wild-type ADPRT activity (a); mutant V455A, kcat = 0.254 +/-0.001, relative to wild-type GH activity (a); relative kcat = 1.00 +/-0.02, wild-type GH activity; relative kcat = 1.00 +/-0.05, wild-type GH activity (a) and (b); relative kcat = 1.00 +/-0.09, wild-type ADPRT activity (A)-(D), and (a) and (b)
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
3-((3,3-diisopropylguanidino)methyl)benzamide
-
37°C, pH 7
0.013
3-((3-(1-benzylpiperidin-4-yl)guanidino)methyl)benzamide
-
37°C, pH 7
0.008
3-((3-(4-bromobenzyl)guanidino)methyl)benzamide
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37°C, pH 7, 1400fold more potent than NAD+ and 400fold more potent than diethylamino(benzylidine-amino)guanidine
0.05
3-((3-benzylguanidino)methyl)benzamide
-
37°C, pH 7
0.071
3-((3-phenethylguanidino)methyl)benzamide
-
37°C, pH 7
0.5
3-((3-tert-butylguanidino)methyl)benzamide
-
37°C, pH 7
0.09
3-(3,3-diisopropylguanidino)benzamide
-
37°C, pH 7
0.5
3-(3-phenylguanidino)benzamide
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37°C, pH 7
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.115
3-((3,3-diisopropylguanidino)methyl)benzamide
Vibrio cholerae
-
37°C, pH 7
0.049
3-((3-(1-benzylpiperidin-4-yl)guanidino)methyl)benzamide
Vibrio cholerae
-
37°C, pH 7
0.031
3-((3-(4-bromobenzyl)guanidino)methyl)benzamide
Vibrio cholerae
-
37°C, pH 7
0.192
3-((3-benzylguanidino)methyl)benzamide
Vibrio cholerae
-
37°C, pH 7
0.272
3-((3-phenethylguanidino)methyl)benzamide
Vibrio cholerae
-
37°C, pH 7
2
3-((3-tert-butylguanidino)methyl)benzamide
Vibrio cholerae
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37°C, pH 7
0.35
3-(3,3-diisopropylguanidino)benzamide
Vibrio cholerae
-
37°C, pH 7
2
3-(3-phenylguanidino)benzamide
Vibrio cholerae
-
37°C, pH 7
0.042
4-amino-1,8-naphthalimide
Vibrio cholerae
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pH 7.9, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.6
-
assay at, nicotinamide + ADPribose-elongation factor 2
7.9
-
assay at
8
-
assay at, NAD+ + elongation factor 2
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
-
polyoma virus transformed baby hamster kidney cells, i.e. pyBHK-cells
Manually annotated by BRENDA team
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-
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Corynebacterium diphtheriae (strain ATCC 27012 / C7 (beta))
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Pseudomonas aeruginosa (strain ATCC 15692 / PAO1 / 1C / PRS 101 / LMG 12228)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Saccharomyces cerevisiae (strain ATCC 204508 / S288c)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
21000
-
SDS-PAGE of purified recombinant fusion enzyme cholera toxin B subunit with glutamic acid carboxylase 65 peptides 531-545 with histidine 6 tag
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 72000, exotoxin A, SDS-PAGE
additional information
-
catalytically active A subunit and five identical B subunits for receptor binding
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
glycoprotein
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-
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
in complex with NAD+ and eEF2, PDB: 3B8H (mutant E546A), 3B82 (mutant E546H), 3B78 (mutant R551H), 2ZIT (wild-type), specific interactions of active-site loop1 with NAD+ and diphthamide results in solvent cover for dinucleotide-binding pocket and coordinates and stabilizes NAD+ in the active-site cleft during ADPRT reaction (transition-state model), crystals are of space group P(1)2(1), C2 symmetry, unit cell parameters: a: 326.9-329.4, b: 68.1-69.2, c: 190.0-191.6, beta: 102.9-103.3, precipitant: PEG-8K or PEG-10K, 6-8%, 1.25 mM NAD+ (in cryo-protection buffer, pH 6.0) soaked into crystals
a 1.8 A crystal structure of cholix in complex with its natural substrate, nicotinamide adenine dinucleotide
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
0.2 microm crossflow microfiltration, concentrated and diafiltered against 10 mM phosphate buffer, pH 7.6, with 30 kDa crossflow ultrafiltration, toxin bound on a S 5/50 GL cation exchange column and eluted with a salt gradient of 10 mM potassium phosphate buffer, pH 7.0 with 1 M NaCl
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adsorption on Ni-NTA resin column, washing and elution with buffer containing 500 mM imidazole, dialyzing in 2 steps
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recombinant catalytic fragment of ExoA, ExoAc
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21 (DE3) with expression vector pET28a
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overexpression of the catalytic fragment of ExoA, ExoAc
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D461A
part of active-site loop 1 (453-463)
D463A
part of active-site loop 1 (453-463)
E546A
reduced ADPRT activity compared to wild-type possibly due to leakage of aqueous solvent into binding pocket which prevents ADP-ribose transfer, no impaired NAD+ binding and glycohydrolase activity, Glu546 crucial for ADPRT activity of ExoA but not strictly conserved among toxin family members, part of active-site loop 3 (546-551), upon NAD+-binding Glu546 and Tyr481 (both connected by hydrogen bonds resulting in water molecule exclusion) in hydrogen bonding distance to nucleophilic N3 of diphthamide imidazole possibly increasing its nucleophilic character
E546A/R551A
double alanine mutant, almost complete loss of ADPRT activity not restored by mutations E546D/R551K or E546R/R551E
E546D
lesser ADPRT activity than E546A
E546F
part of active-site loop 3 (546-551)
E546N
partial rescue of ADPRT activity compared to E546A
E546Q
part of active-site loop 3 (546-551)
E547A
part of active-site loop 3 (546-551)
E548A
part of active-site loop 3 (546-551)
E553A
impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members
G453A
part of active-site loop 1 (453-463)
G454A
part of active-site loop 1 (453-463); reduced ADPRT activity compared to wild-type, Gly454 is part of active-site loop 1 (453-463)
G549A
part of active-site loop 3 (546-551)
G550A
part of active-site loop 3 (546-551)
L462A
part of active-site loop 1 (453-463)
L552A
QuickChange mutagenesis
Q460A
reduced ADPRT activity compared to wild-type, Gln460 is part of active-site loop 1 (453-463), active-site loop1 flips towards diphthamide of eEF2 by a hinged action of Ala457 and Ala464 upon NAD+ binding which places Gln460 close to adenine phosphate of NAD+, interaction of Gln460 with A-phosphate of NAD+ possibly prevents hydrolysis of NAD+ to AMP or ADP
R456A
part of active-site loop 1 (453-463)
R458A
56-fold reduction in ADPRT activity, 53-fold reduction in GH activity, and 31-fold increased KD for NAD+ compared to wild-type, ADPRT activity sensitivity towards substitution in order Gln>Lys>Trp>Ala>His, Arg458 is part of active-site loop 1 (453-463) and implicated in NAD+ substrate docking and orientation, active-site loop1 flips towards diphthamide of eEF2 by a hinged action of Ala457 and Ala464 upon NAD+ binding which enables van der Waals interactions between Arg458 and the adenine base of NAD+
R458H
part of active-site loop 1 (453-463)
R458K
part of active-site loop 1 (453-463)
R458Q
part of active-site loop 1 (453-463)
R458W
part of active-site loop 1 (453-463)
R551A
ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)
R551C
part of active-site loop 3 (546-551)
R551E
part of active-site loop 3 (546-551)
R551H
part of active-site loop 3 (546-551)
R551K
part of active-site loop 3 (546-551)
R551Q
part of active-site loop 3 (546-551)
S459A
part of active-site loop 1 (453-463)
V455A
part of active-site loop 1 (453-463)
DELTA424-634
-
catalytic domain of cholix has much greater affinity for NAD+ than the full-length enzyme. 15fold higher ADP-ribosyltransferase activity than full-length enzyme
E574A
-
mutation of catalytic fragment DELTA424-634: mutant does not have a large impact on NAD+-binding. Catalytic activity is 3fold reduced compared to catalytic fragment DELTA424-634
E574A/E581A
-
mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity
E579R
-
mutation of catalytic fragment DELTA424-634: NAD+ binding not changed significantly. Catalytic activity is 1.6fold reduced compared to catalytic fragment DELTA424-634
E581A
-
mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity
Y493A
-
mutation of catalytic fragment DELTA424-634: mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant shows a marked reduction in activity compared to catalytic fragment DELTA424-634
Y504A
-
mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant shows a marked reduction in activity compared to catalytic fragment DELTA424-634
Y504F
-
mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant has lower affinity for NAD+ than Y504A mutant. Mutant shows a mild reduction in activity compared to catalytic fragment DELTA424-634
additional information
-
recombinant gene of cholera toxin B subunit with triple glutamic acid decarboxylase 65 peptides 531-545 (3p531) with 6-bp oligonucleotide sequences as flexible hinges
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
diagnostics
drug development
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
medicine
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therapy for autoimmune diabetes with glutamic acid decarboxylase 65 as autoantigen combined with cholera toxin B subunit as adjuvans lowering the number of antigens needed for autoimmune suppression, oral application for 5 weeks reduces insulitis of pancreas in mouse model