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 ACCESSION NO.
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 ACCESSION NO.
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 ACCESSION NO.
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
SEQUENCE CODE
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 ACCESSION NO.
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 ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
4
-
diethylamino(benzylidine-amino)guanidine
-
-
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]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
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]
Ki VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
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]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
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
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.6
-
-
assay at, nicotinamide + ADPribose-elongation factor 2
7.9
-
-
assay at
8
-
-
assay at, NAD+ + elongation factor 2
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
22
-
-
assay at
25
-
-
assay at
25
-
-
assay at
SOURCE TISSUE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
-
polyoma virus transformed baby hamster kidney cells, i.e. pyBHK-cells
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
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)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
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 ACCESSION NO.
COMMENTARY
LITERATURE
monomer
-
1 * 72000, exotoxin A, SDS-PAGE
additional information
-
catalytically active A subunit and five identical B subunits for receptor binding
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
glycoprotein
-
-
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
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
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant catalytic fragment of ExoA, ExoAc
-
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
-
adsorption on Ni-NTA resin column, washing and elution with buffer containing 500 mM imidazole, dialyzing in 2 steps
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
overexpression of the catalytic fragment of ExoA, ExoAc
-
expressed in Escherichia coli BL21 (DE3) with expression vector pET28a
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ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
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
-
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
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
E581A
-
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 ACCESSION NO.
COMMENTARY
LITERATURE
drug development
P11439
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
diagnostics
-
toxin purification and recognition to enable cost effective toxin free cholera vaccine production, detection minimum 1 ng/ml
medicine
-
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
-
toxin purification and recognition to enable cost effective toxin free cholera vaccine production, detection minimum 1 ng/ml
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