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
2.4.2.36
-
RECOMMENDED NAME
GeneOntology No.
NAD+-diphthamide ADP-ribosyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY
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
LITERATURE
pentosyl group transfer
-
-
-
-
pentosyl group transfer
P11439
-
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.
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
(adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotide-elongation factor 2
-
-
-
-
ADP-ribosyltransferase
-
-
-
-
cholera toxin
-
-
-
cholix
-
-
CTB
-
cholera toxin B subunit
ExoA(c)
P11439
-
exotoxin A
P11439
-
mono(ADPribosyl)transferase
-
-
-
-
mono-ADP-ribosyltransferase
-
-
NAD(+)-diphthamide ADP-ribosyltransferase
-
-
-
-
NAD-diphthamide ADP-ribosyltransferase
-
-
-
-
NAD-diphthamide ADP-ribosyltransferase NAD-elongation factor 2 ADP-ribosyltransferase
-
-
-
-
NAD:elongation factor 2-adenosine diphosphate ribose-transferase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
52933-21-8
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain 569B, hyper-producer of cholera toxin
-
-
Manually annotated by BRENDA team
strain 569B, hyper-producer of cholera toxin
-
-
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
diethylamino(benzylidine-amino)guanidine + NAD+
?
show the reaction diagram
-
DEABAG
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
-
-
-
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
-
-
r
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
-
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
liver enzyme
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
NAD-glycohydrolase activity without acceptor substrate
-
-
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
i.e. elongation factor 2 of baby hamster kidney cells, forward ADP-ribosylation reaction is reversed by fragment A from Pseudomonas aeruginosa
-
r
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
reaction involves a random-order ternary complex
-
-
?
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), essential ribosomal elongation factor 2: eEF2, 2-step reaction by concerted GH and ADPRT activity, transition-state model
-
-
?
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
-
-
-
-
?
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, i.e. eukaryotic elongation factor 2 with a diphthamide-containing loop comprising residues Leu693-Gly703, ADP-ribosylation at His699, site determination by mass spectrometry and mutational analysis, overview
-
-
?
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, no ADP-ribosyl transfer by mutants R551H and R551C despite of occurring NAD+-binding and hydrolysis
-
-
-
additional information
?
-
-
cholera toxin activates mouse bone marrow mast cells to release interleukin-4 via activation of nucleotide oligomerisation domain 1 and toll-like receptor-4, and initiation of antigen-specific T-helper 2 response
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
-
-
-
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
-
-
r
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
liver enzyme
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
NAD-glycohydrolase activity without acceptor substrate
-
-
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
show the reaction diagram
-
i.e. elongation factor 2 of baby hamster kidney cells, forward ADP-ribosylation reaction is reversed by fragment A from Pseudomonas aeruginosa
-
r
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)
-
-
?
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
-
-
-
-
?
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
-
-
?
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
-
-
-
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
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
-
1 mM, 37°C, pH 7, 68% inhibition
3-((3-(2-(ethyl(m-tolyl)amino)ethyl)guanidino)methyl)benzamide
-
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
-
1 mM, 37°C, pH 7, 98% inhibition
3-((3-(4-methoxybenzyl)guanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 97% inhibition, not sufficient material for IC50 and Ki estimation
3-((3-benzylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 93% inhibition
3-((3-phenethylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 93% inhibition
3-((3-phenylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 57% inhibition
3-((3-tert-butylguanidino)methyl)benzamide
-
1 mM, 37°C, pH 7, 39% inhibition
3-(3,3-diisopropylguanidino)benzamide
-
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
-
1 mM, 37°C, pH 7, 10% inhibition
3-(3-phenylguanidino)benzamide
-
1 mM, 37°C, pH 7, 12% inhibition
3-(3-tert-butylguanidino)benzamide
-
1 mM, 37°C, pH 7, 77% inhibition
4-amino-1,8-naphthalimide
-
-
Cytoplasmic extract of pyBHK-cells
-
not fragment A
-
Elastase
-
inactivation follows pseudo-first order kinetic
-
ethyl 4-(3-(3-carbamoylbenzyl)guanidino)piperidine-1-carboxylate
-
1 mM, 37°C, pH 7, 34% inhibition
histamine
-
250 mM, almost complete inhibition
additional information
-
cellular ADPribosyltransferase is not inhibited by anti-fragment A-antiserum
-
additional information
-
fragment A and toxin A are not inhibited by histamine
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-mercaptoethanol
-
activation, together with SDS or guanidine hydrochloride
cysteine
-
activation, together with SDS or guanidine hydrochloride
dithiothreitol
-
enhances activity together with urea
SDS
-
enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite
sulfite
-
activation, together with SDS or guanidine hydrochloride
Urea
-
enhances activity together with dithiothreitol
guanidine hydrochloride
-
enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite
additional information
-
exotoxin is synthesized in a catalytically inactive, proenzyme form, unfolding of the toxin molecule may cause activation
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
4
diethylamino(benzylidine-amino)guanidine
-
-
14
NAD+
-
-
0.02
elongation factor 2
-
-
additional information
NAD+
P11439
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
LITERATURE
IMAGE
12
elongation factor 2
-
-
additional information
NAD+
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
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
-
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
-
37°C, pH 7
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.115
3-((3,3-diisopropylguanidino)methyl)benzamide
-
37°C, pH 7
0.049
3-((3-(1-benzylpiperidin-4-yl)guanidino)methyl)benzamide
-
37°C, pH 7
0.031
3-((3-(4-bromobenzyl)guanidino)methyl)benzamide
-
37°C, pH 7
0.192
3-((3-benzylguanidino)methyl)benzamide
-
37°C, pH 7
0.272
3-((3-phenethylguanidino)methyl)benzamide
-
37°C, pH 7
2
3-((3-tert-butylguanidino)methyl)benzamide
-
37°C, pH 7
0.35
3-(3,3-diisopropylguanidino)benzamide
-
37°C, pH 7
2
3-(3-phenylguanidino)benzamide
-
37°C, pH 7
0.042
4-amino-1,8-naphthalimide
-
pH 7.9, 25C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
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
LITERATURE
25
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
-
polyoma virus transformed baby hamster kidney cells, i.e. pyBHK-cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
fragment A of diphtheria toxin
-
Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
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
LITERATURE
21000
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SDS-PAGE of purified recombinant fusion enzyme cholera toxin B subunit with glutamic acid carboxylase 65 peptides 531-545 with histidine 6 tag
701050
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
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1 * 72000, exotoxin A, SDS-PAGE
additional information
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catalytically active A subunit and five identical B subunits for receptor binding
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
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
P11439
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
recombinant catalytic fragment of ExoA, ExoAc
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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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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
overexpression of the catalytic fragment of ExoA, ExoAc
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expressed in Escherichia coli BL21 (DE3) with expression vector pET28a
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D461A
P11439
part of active-site loop 1 (453-463)
D463A
P11439
part of active-site loop 1 (453-463)
E546A
P11439
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
P11439
double alanine mutant, almost complete loss of ADPRT activity not restored by mutations E546D/R551K or E546R/R551E
E546D
P11439
lesser ADPRT activity than E546A
E546F
P11439
part of active-site loop 3 (546-551)
E546H
P11439
see E546A
E546N
P11439
partial rescue of ADPRT activity compared to E546A
E546Q
P11439
part of active-site loop 3 (546-551)
E547A
P11439
part of active-site loop 3 (546-551)
E548A
P11439
part of active-site loop 3 (546-551)
E553A
P11439
impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members
G453A
P11439
part of active-site loop 1 (453-463)
G454A
P11439
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
P11439
part of active-site loop 3 (546-551)
G550A
P11439
part of active-site loop 3 (546-551)
L462A
P11439
part of active-site loop 1 (453-463)
L552A
P11439
QuickChange mutagenesis
Q460A
P11439
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
P11439
part of active-site loop 1 (453-463)
R458A
P11439
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
P11439
part of active-site loop 1 (453-463)
R458K
P11439
part of active-site loop 1 (453-463)
R458Q
P11439
part of active-site loop 1 (453-463)
R458W
P11439
part of active-site loop 1 (453-463)
R551A
P11439
ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)
R551C
P11439
part of active-site loop 3 (546-551)
R551E
P11439
part of active-site loop 3 (546-551)
R551H
P11439
part of active-site loop 3 (546-551)
R551K
P11439
part of active-site loop 3 (546-551)
R551Q
P11439
part of active-site loop 3 (546-551)
S459A
P11439
part of active-site loop 1 (453-463)
V455A
P11439
part of active-site loop 1 (453-463)
DELTA424-634
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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
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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
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mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity
E579R
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mutation of catalytic fragment DELTA424-634: NAD+ binding not changed significantly. Catalytic activity is 1.6fold reduced compared to catalytic fragment DELTA424-634
Y493A
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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
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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
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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
E581A
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mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity
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
LITERATURE
drug development
P11439
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
diagnostics
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toxin purification and recognition to enable cost effective toxin free cholera vaccine production, detection minimum 1 ng/ml
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
diagnostics
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toxin purification and recognition to enable cost effective toxin free cholera vaccine production, detection minimum 1 ng/ml
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