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Information on EC 2.4.2.36 - NAD+-diphthamide ADP-ribosyltransferase and Organism(s) Pseudomonas aeruginosa and UniProt Accession P11439
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2.4.2.36 NAD+-diphthamide ADP-ribosyltransferaseIUBMB Comments
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.
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Word Map
- 2.4.2.36
- camp
- cyclase
- vibrio
- mucosal
- pertussis
-
adp-ribosylation
- adenylate
- forskolin
-
retrograde
- gm1
- ganglioside
- aeruginosa
- enterotoxin
- iga
- agonist
-
tracer
-
dorsal
- prostaglandin
-
heat-labile
-
intranasal
-
dibutyryl
-
afferent
- isoproterenol
-
adenylyl
- g-proteins
-
innervation
-
gtp-binding
- diphtheria
- immunotoxins
-
anterograde
-
b-subunits
-
rostrally
-
toxigenic
-
ribosylation
- pilus
-
toxin-induced
-
toxin-sensitive
-
pyrogenic
- toxoid
- 8-bromo-camp
-
diarrheal
- antitoxin
-
ventrolateral
-
peyer
-
exoenzyme
-
enterotoxigenic
-
gppnhp
-
preganglionic
- drug development
-
wga-hrp
-
parabrachial
- medicine
- diagnostics
The taxonomic range for the selected organisms is: Pseudomonas aeruginosa
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Reaction Schemes
+
=
+
+
=
+
N-(ADP-D-ribosyl)diphthamide-[translation elongation factor 2]
Synonyms
cholera toxin, exotoxin a, adp-ribosyltransferase, mono-adp-ribosyltransferase, cholix toxin, cholix, ehtoxin-like, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
(adenosine diphosphoribose)transferase, nicotinamide adenine dinucleotide-elongation factor 2
-
-
-
-
ADP-ribosyltransferase
-
-
-
-
mono(ADPribosyl)transferase
-
-
-
-
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
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
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
52933-21-8
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NAD+ + diphthamide-[translation elongation factor 2]
nicotinamide + N-(ADP-D-ribosyl)diphthamide-[translation elongation factor 2]
-
-
-
?
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
NAD+ + peptide diphthamide
nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide
-
-
-
-
?
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
transfer of ADP-ribose moiety (oxocarbenium ion) of NAD+ onto N3 of diphthamide imidazole of essential ribosomal elongation factor 2 (eEF2)
-
-
?
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
essential ribosomal elongation factor 2: eEF2, 2-step reaction by concerted GH and ADPRT activity, transition-state model
-
-
?
eEF2 + NAD+
eEF2 N-(ADP-D-ribosyl)diphthamide + NADH + H+
-
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
-
-
?
eEF2 + NAD+
eEF2 N-(ADP-D-ribosyl)diphthamide + NADH + H+
-
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
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
-
-
r
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
NAD-glycohydrolase activity without acceptor substrate
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
reaction involves a random-order ternary complex
-
-
?
additional information
?
-
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
-
-
?
additional information
?
-
-
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
-
-
?
additional information
?
-
no ADP-ribosyl transfer by mutants R551H and R551C despite of occurring NAD+-binding and hydrolysis
-
-
?
additional information
?
-
-
no ADP-ribosyl transfer by mutants R551H and R551C despite of occurring NAD+-binding and hydrolysis
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NAD+ + diphthamide-[translation elongation factor 2]
nicotinamide + N-(ADP-D-ribosyl)diphthamide-[translation elongation factor 2]
-
-
-
?
NAD+ + essential ribosomal elongation factor 2
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
transfer of ADP-ribose moiety (oxocarbenium ion) of NAD+ onto N3 of diphthamide imidazole of essential ribosomal elongation factor 2 (eEF2)
-
-
?
eEF2 + NAD+
eEF2 N-(ADP-D-ribosyl)diphthamide + NADH + H+
-
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
-
-
?
NAD+ + peptide diphthamide
nicotinamide + peptide N-(ADP-D-ribosyl)diphthamide
-
-
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
-
-
?
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
-
-
r
NAD+ + elongation factor 2
nicotinamide + ADPribose-elongation factor 2
-
NAD-glycohydrolase activity without acceptor substrate
-
?
additional information
?
-
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
-
-
?
additional information
?
-
-
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
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
2-[(5,6-dimethyl-4-oxo-3,4,4a,7a-tetrahydrothieno[2,3-d]pyrimidin-2-yl)sulfanyl]-N-(2-hydroxyethyl)acetamide
-
8-fluoro-2-[3-(piperidin-1-yl)propanoyl]-1,3,4,5-tetrahydrobenzo[c][1,6]naphthyridin-6(2H)-one
-
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
guanidine hydrochloride
-
enhances activity together with dithiothreitol, cysteine, 2-mercaptoethanol or sulfite
SDS
-
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
additional information
NAD+
double mutant E546A/R551A, relative KD = 1.11 +/-0.03
additional information
NAD+
-
double mutant E546A/R551A, relative KD = 1.11 +/-0.03
additional information
NAD+
double mutant E546D/R551K, relative KD = 2.71 +/-0.74
additional information
NAD+
-
double mutant E546D/R551K, relative KD = 2.71 +/-0.74
additional information
NAD+
double mutant E546R/R551E, relative KD = 1.29 +/-0.49
additional information
NAD+
-
double mutant E546R/R551E, relative KD = 1.29 +/-0.49
additional information
NAD+
mutant D461A, KD = 1.03 +/-0.02 relative to wild-type (c)
additional information
NAD+
-
mutant D461A, KD = 1.03 +/-0.02 relative to wild-type (c)
additional information
NAD+
mutant D463A, KD = 1.18 +/-0.04 relative to wild-type (c)
additional information
NAD+
-
mutant D463A, KD = 1.18 +/-0.04 relative to wild-type (c)
additional information
NAD+
mutant E546A, relative KD = 1.77 +/-0.34
additional information
NAD+
-
mutant E546A, relative KD = 1.77 +/-0.34
additional information
NAD+
mutant E546D, relative KD = 3.83 +/-0.89
additional information
NAD+
-
mutant E546D, relative KD = 3.83 +/-0.89
additional information
NAD+
mutant E546F, relative KD = 1.97 +/-0.11
additional information
NAD+
-
mutant E546F, relative KD = 1.97 +/-0.11
additional information
NAD+
mutant E546H, relative KD = 1.03 +/-0.09
additional information
NAD+
-
mutant E546H, relative KD = 1.03 +/-0.09
additional information
NAD+
mutant E546N, relative KD = 2.14 +/-0.26
additional information
NAD+
-
mutant E546N, relative KD = 2.14 +/-0.26
additional information
NAD+
mutant E546Q, relative KD = 2.31 +/-0.71
additional information
NAD+
-
mutant E546Q, relative KD = 2.31 +/-0.71
additional information
NAD+
mutant E547A, relative KD = 1.97 +/-0.77
additional information
NAD+
-
mutant E547A, relative KD = 1.97 +/-0.77
additional information
NAD+
mutant E548A, relative KD = 2.69 +/-0.11
additional information
NAD+
-
mutant E548A, relative KD = 2.69 +/-0.11
additional information
NAD+
mutant E553A, relative KD = 2.57 +/-0.86
additional information
NAD+
-
mutant E553A, relative KD = 2.57 +/-0.86
additional information
NAD+
mutant G453A, KD = 4.85 +/-1.07 relative to wild-type (c)
additional information
NAD+
-
mutant G453A, KD = 4.85 +/-1.07 relative to wild-type (c)
additional information
NAD+
mutant G454A, KD = 6.25 +/-0.22 relative to wild-type (c)
additional information
NAD+
-
mutant G454A, KD = 6.25 +/-0.22 relative to wild-type (c)
additional information
NAD+
mutant G549A, relative KD = 8.00 +/-0.94
additional information
NAD+
-
mutant G549A, relative KD = 8.00 +/-0.94
additional information
NAD+
mutant G550A, relative KD = 6.60 +/-0.54
additional information
NAD+
-
mutant G550A, relative KD = 6.60 +/-0.54
additional information
NAD+
mutant L462A, KD = 1.86 +/-0.10 relative to wild-type (c)
additional information
NAD+
-
mutant L462A, KD = 1.86 +/-0.10 relative to wild-type (c)
additional information
NAD+
mutant L552A, relative KD = 4.43 +/-0.57
additional information
NAD+
-
mutant L552A, relative KD = 4.43 +/-0.57
additional information
NAD+
mutant Q460A, KD = 4.30 +/-0.43 relative to wild-type (c)
additional information
NAD+
-
mutant Q460A, KD = 4.30 +/-0.43 relative to wild-type (c)
additional information
NAD+
mutant R456A, KD = 3.56 +/-0.53 relative to wild-type (c)
additional information
NAD+
-
mutant R456A, KD = 3.56 +/-0.53 relative to wild-type (c)
additional information
NAD+
mutant R458A, KD = 5.78 +/-0.18 relative to wild-type (c)
additional information
NAD+
-
mutant R458A, KD = 5.78 +/-0.18 relative to wild-type (c)
additional information
NAD+
mutant R458H, KD = 31.02 +/-7.18 relative to wild-type (d)
additional information
NAD+
-
mutant R458H, KD = 31.02 +/-7.18 relative to wild-type (d)
additional information
NAD+
mutant R458K, KD = 3.18 +/-0.23 relative to wild-type (c)
additional information
NAD+
-
mutant R458K, KD = 3.18 +/-0.23 relative to wild-type (c)
additional information
NAD+
mutant R458Q, KD = 0.45 +/-0.06 relative to wild-type (e)
additional information
NAD+
-
mutant R458Q, KD = 0.45 +/-0.06 relative to wild-type (e)
additional information
NAD+
mutant R458W, KD = 11.26 +/-1.16 relative to wild-type (d)
additional information
NAD+
-
mutant R458W, KD = 11.26 +/-1.16 relative to wild-type (d)
additional information
NAD+
mutant R551A, relative KD = 2.69 +/-0.03
additional information
NAD+
-
mutant R551A, relative KD = 2.69 +/-0.03
additional information
NAD+
mutant R551C, relative KD = 5.57 +/-0.91
additional information
NAD+
-
mutant R551C, relative KD = 5.57 +/-0.91
additional information
NAD+
mutant R551E, relative KD = 5.03 +/-0.51
additional information
NAD+
-
mutant R551E, relative KD = 5.03 +/-0.51
additional information
NAD+
mutant R551H, relative KD = 2.29 +/-0.46
additional information
NAD+
-
mutant R551H, relative KD = 2.29 +/-0.46
additional information
NAD+
mutant R551K, relative KD = 1.29 +/-0.03
additional information
NAD+
-
mutant R551K, relative KD = 1.29 +/-0.03
additional information
NAD+
mutant R551Q, relative KD = 2.63 +/-0.39
additional information
NAD+
-
mutant R551Q, relative KD = 2.63 +/-0.39
additional information
NAD+
mutant S459A, KD = 1.59 +/-0.16 relative to wild-type (c)
additional information
NAD+
-
mutant S459A, KD = 1.59 +/-0.16 relative to wild-type (c)
additional information
NAD+
mutant V455A, KD = 3.31 +/-0.24 relative to wild-type (c)
additional information
NAD+
-
mutant V455A, KD = 3.31 +/-0.24 relative to wild-type (c)
additional information
NAD+
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
additional information
NAD+
-
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
additional information
NAD+
wild-type, KD = 36 +/-8 microM (c), KD = 62 +/-2 microM (d), KD = 74 +/-16 microM, relative KD = 1.00 +/-0.03
additional information
NAD+
-
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
additional information
NAD+
0.0062-0.13/min, wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
-
0.0062-0.13/min, wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
0.125 +/-0.007/min (a), wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
-
0.125 +/-0.007/min (a), wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
0.130 +/-0.012/min (b), wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
-
0.130 +/-0.012/min (b), wild-type GH activity, in the absence of eEF2, pH 7.9, 25°C, 60 min
additional information
NAD+
1306 +/-121/min (a), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
1306 +/-121/min (a), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
1495 +/-224/min (b), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
1495 +/-224/min (b), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
628 +/-8/min (B), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
628 +/-8/min (B), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
746 +/-18/min (A), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
746 +/-18/min (A), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
847 +/-21/min (C), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
847 +/-21/min (C), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
900 +/-20/min (D), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
-
900 +/-20/min (D), wild-type ADPRT activity, 12 microM eEF2, pH 7.9, 25°C, 300 sec
additional information
NAD+
double mutant E546A/R551A, kcat = 0.0001 +/-0.0001, relative to wild-type ADPRT activity (C)
additional information
NAD+
-
double mutant E546A/R551A, kcat = 0.0001 +/-0.0001, relative to wild-type ADPRT activity (C)
additional information
NAD+
double mutant E546A/R551A, kcat = 0.84 +/-0.04, relative to wild-type GH activity
additional information
NAD+
-
double mutant E546A/R551A, kcat = 0.84 +/-0.04, relative to wild-type GH activity
additional information
NAD+
double mutant E546D/R551K, kcat = 0.0009 +/-0.0013, relative to wild-type ADPRT activity (C)
additional information
NAD+
-
double mutant E546D/R551K, kcat = 0.0009 +/-0.0013, relative to wild-type ADPRT activity (C)
additional information
NAD+
double mutant E546D/R551K, kcat = 0.42 +/-0.0002, relative to wild-type GH activity
additional information
NAD+
-
double mutant E546D/R551K, kcat = 0.42 +/-0.0002, relative to wild-type GH activity
additional information
NAD+
double mutant E546R/R551E, kcat = 0.00001 +/-0.000002, relative to wild-type ADPRT activity (C)
additional information
NAD+
-
double mutant E546R/R551E, kcat = 0.00001 +/-0.000002, relative to wild-type ADPRT activity (C)
additional information
NAD+
double mutant E546R/R551E, kcat = 0.38 +/-0.01, relative to wild-type GH activity
additional information
NAD+
-
double mutant E546R/R551E, kcat = 0.38 +/-0.01, relative to wild-type GH activity
additional information
NAD+
mutant D461A, kcat = 1.001 +/-0.091, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant D461A, kcat = 1.001 +/-0.091, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant D461A, kcat = 1.076 +/-0.041, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant D461A, kcat = 1.076 +/-0.041, relative to wild-type GH activity (a)
additional information
NAD+
mutant D463A, kcat = 1.148 +/-0.038, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant D463A, kcat = 1.148 +/-0.038, relative to wild-type GH activity (a)
additional information
NAD+
mutant D463A, kcat = 1.270 +/-0.204, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant D463A, kcat = 1.270 +/-0.204, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant E546A, kcat = 0.0012 +/-0.00002, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant E546A, kcat = 0.0012 +/-0.00002, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant E546A, kcat = 0.24 +/-0.008, relative to wild-type GH activity
additional information
NAD+
-
mutant E546A, kcat = 0.24 +/-0.008, relative to wild-type GH activity
additional information
NAD+
mutant E546D, kcat = 0.00025 +/-0.00005, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant E546D, kcat = 0.00025 +/-0.00005, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant E546D, kcat = 0.45 +/-0.002, relative to wild-type GH activity
additional information
NAD+
-
mutant E546D, kcat = 0.45 +/-0.002, relative to wild-type GH activity
additional information
NAD+
mutant E546F, kcat = 0.007 +/-0.0002, relative to wild-type ADPRT activity (D)
additional information
NAD+
-
mutant E546F, kcat = 0.007 +/-0.0002, relative to wild-type ADPRT activity (D)
additional information
NAD+
mutant E546F, kcat = 0.69 +/-0.05, relative to wild-type GH activity
additional information
NAD+
-
mutant E546F, kcat = 0.69 +/-0.05, relative to wild-type GH activity
additional information
NAD+
mutant E546H, kcat = 0.0012 +/-0.0002, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant E546H, kcat = 0.0012 +/-0.0002, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant E546H, kcat = 0.59 +/-0.009, relative to wild-type GH activity
additional information
NAD+
-
mutant E546H, kcat = 0.59 +/-0.009, relative to wild-type GH activity
additional information
NAD+
mutant E546N, kcat = 0.0010 +/-0.00009, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant E546N, kcat = 0.0010 +/-0.00009, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant E546N, kcat = 0.67 +/-0.03, relative to wild-type GH activity
additional information
NAD+
-
mutant E546N, kcat = 0.67 +/-0.03, relative to wild-type GH activity
additional information
NAD+
mutant E546Q, kcat = 0.011 +/-0.002, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant E546Q, kcat = 0.011 +/-0.002, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant E546Q, kcat = 0.80 +/-0.04, relative to wild-type GH activity
additional information
NAD+
-
mutant E546Q, kcat = 0.80 +/-0.04, relative to wild-type GH activity
additional information
NAD+
mutant E547A, kcat = 0.795 +/-0.077, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant E547A, kcat = 0.795 +/-0.077, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant E547A, kcat = 0.83 +/-0.14, relative to wild-type GH activity
additional information
NAD+
-
mutant E547A, kcat = 0.83 +/-0.14, relative to wild-type GH activity
additional information
NAD+
mutant E548A, kcat = 0.76 +/-0.01, relative to wild-type GH activity
additional information
NAD+
-
mutant E548A, kcat = 0.76 +/-0.01, relative to wild-type GH activity
additional information
NAD+
mutant E548A, kcat = 1.669 +/-0.298, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant E548A, kcat = 1.669 +/-0.298, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant E553A, kcat = 0.0015 +/-0.00005, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant E553A, kcat = 0.0015 +/-0.00005, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant E553A, kcat = 0.07 +/-0.009, relative to wild-type GH activity
additional information
NAD+
-
mutant E553A, kcat = 0.07 +/-0.009, relative to wild-type GH activity
additional information
NAD+
mutant G453A, kcat = 0.290 +/-0.040, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant G453A, kcat = 0.290 +/-0.040, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant G453A, kcat = 0.331 +/-0.019, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant G453A, kcat = 0.331 +/-0.019, relative to wild-type GH activity (a)
additional information
NAD+
mutant G454A, kcat = 0.031 +/-0.003, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant G454A, kcat = 0.031 +/-0.003, relative to wild-type GH activity (a)
additional information
NAD+
mutant G454A, kcat = 0.046 +/-0.008, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant G454A, kcat = 0.046 +/-0.008, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant G549A, kcat = 0.30 +/-0.005, relative to wild-type GH activity
additional information
NAD+
-
mutant G549A, kcat = 0.30 +/-0.005, relative to wild-type GH activity
additional information
NAD+
mutant G549A, kcat = 0.770 +/-0.076, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant G549A, kcat = 0.770 +/-0.076, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant G550A, kcat = 0.41 +/-0.04, relative to wild-type GH activity
additional information
NAD+
-
mutant G550A, kcat = 0.41 +/-0.04, relative to wild-type GH activity
additional information
NAD+
mutant G550A, kcat = 0.562 +/-0.166, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant G550A, kcat = 0.562 +/-0.166, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant L462A, kcat = 0.268 +/-0.041, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant L462A, kcat = 0.268 +/-0.041, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant L462A, kcat = 0.955 +/-0.015, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant L462A, kcat = 0.955 +/-0.015, relative to wild-type GH activity (a)
additional information
NAD+
mutant L552A, kcat = 0.58 +/-0.002, relative to wild-type GH activity
additional information
NAD+
-
mutant L552A, kcat = 0.58 +/-0.002, relative to wild-type GH activity
additional information
NAD+
mutant L552A, kcat = 2.365 +/-0.527, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant L552A, kcat = 2.365 +/-0.527, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant Q460A, kcat = 0.115 +/-0.010, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant Q460A, kcat = 0.115 +/-0.010, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant Q460A, kcat = 0.622 +/-0.014, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant Q460A, kcat = 0.622 +/-0.014, relative to wild-type GH activity (a)
additional information
NAD+
mutant R456A, kcat = 0.230 +/-0.010, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant R456A, kcat = 0.230 +/-0.010, relative to wild-type GH activity (a)
additional information
NAD+
mutant R456A, kcat = 0.239 +/-0.034, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant R456A, kcat = 0.239 +/-0.034, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant R458A, kcat = 0.132 +/-0.031, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant R458A, kcat = 0.132 +/-0.031, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant R458A, kcat = 0.249 +/-0.021, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant R458A, kcat = 0.249 +/-0.021, relative to wild-type GH activity (a)
additional information
NAD+
mutant R458H, kcat = 0.018 +/-0.003, relative to wild-type GH activity (b)
additional information
NAD+
-
mutant R458H, kcat = 0.018 +/-0.003, relative to wild-type GH activity (b)
additional information
NAD+
mutant R458H, kcat = 0.019 +/-0.007, relative to wild-type ADPRT activity (b)
additional information
NAD+
-
mutant R458H, kcat = 0.019 +/-0.007, relative to wild-type ADPRT activity (b)
additional information
NAD+
mutant R458K, kcat = 0.428 +/-0.033, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant R458K, kcat = 0.428 +/-0.033, relative to wild-type GH activity (a)
additional information
NAD+
mutant R458K, kcat = 0.543 +/-0.155, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant R458K, kcat = 0.543 +/-0.155, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant R458Q, kcat = 0.650 +/-0.130, relative to wild-type ADPRT activity (b)
additional information
NAD+
-
mutant R458Q, kcat = 0.650 +/-0.130, relative to wild-type ADPRT activity (b)
additional information
NAD+
mutant R458Q, kcat = 0.991 +/-0.017, relative to wild-type GH activity (b)
additional information
NAD+
-
mutant R458Q, kcat = 0.991 +/-0.017, relative to wild-type GH activity (b)
additional information
NAD+
mutant R458W, kcat = 0.379 +/-0.043, relative to wild-type ADPRT activity (b)
additional information
NAD+
-
mutant R458W, kcat = 0.379 +/-0.043, relative to wild-type ADPRT activity (b)
additional information
NAD+
mutant R458W, kcat = 0.438 +/-0.040, relative to wild-type GH activity (b)
additional information
NAD+
-
mutant R458W, kcat = 0.438 +/-0.040, relative to wild-type GH activity (b)
additional information
NAD+
mutant R551A, kcat = 0.32 +/-0.005, relative to wild-type GH activity
additional information
NAD+
-
mutant R551A, kcat = 0.32 +/-0.005, relative to wild-type GH activity
additional information
NAD+
mutant R551A, kcat = 0.380 +/-0.024, relative to wild-type ADPRT activity (A)
additional information
NAD+
-
mutant R551A, kcat = 0.380 +/-0.024, relative to wild-type ADPRT activity (A)
additional information
NAD+
mutant R551C, kcat = ~0, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant R551C, kcat = ~0, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant R551C, kcat = 0.10 +/-0.006, relative to wild-type GH activity
additional information
NAD+
-
mutant R551C, kcat = 0.10 +/-0.006, relative to wild-type GH activity
additional information
NAD+
mutant R551E, kcat = 0.011 +/-0.005, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant R551E, kcat = 0.011 +/-0.005, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant R551E, kcat = 0.09 +/-0.001, relative to wild-type GH activity
additional information
NAD+
-
mutant R551E, kcat = 0.09 +/-0.001, relative to wild-type GH activity
additional information
NAD+
mutant R551H, kcat = ~0, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant R551H, kcat = ~0, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant R551H, kcat = 0.41 +/-0.002, relative to wild-type GH activity
additional information
NAD+
-
mutant R551H, kcat = 0.41 +/-0.002, relative to wild-type GH activity
additional information
NAD+
mutant R551K, kcat = 0.317 +/-0.024, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant R551K, kcat = 0.317 +/-0.024, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant R551K, kcat = 0.69 +/-0.03, relative to wild-type GH activity
additional information
NAD+
-
mutant R551K, kcat = 0.69 +/-0.03, relative to wild-type GH activity
additional information
NAD+
mutant R551Q, kcat = 0.185 +/-0.014, relative to wild-type ADPRT activity (B)
additional information
NAD+
-
mutant R551Q, kcat = 0.185 +/-0.014, relative to wild-type ADPRT activity (B)
additional information
NAD+
mutant R551Q, kcat = 0.38 +/-0.01, relative to wild-type GH activity
additional information
NAD+
-
mutant R551Q, kcat = 0.38 +/-0.01, relative to wild-type GH activity
additional information
NAD+
mutant S459A, kcat = 1.119 +/-0.054, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant S459A, kcat = 1.119 +/-0.054, relative to wild-type GH activity (a)
additional information
NAD+
mutant S459A, kcat = 1.256 +/-0.144, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant S459A, kcat = 1.256 +/-0.144, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant V455A, kcat = 0.241 +/-0.032, relative to wild-type ADPRT activity (a)
additional information
NAD+
-
mutant V455A, kcat = 0.241 +/-0.032, relative to wild-type ADPRT activity (a)
additional information
NAD+
mutant V455A, kcat = 0.254 +/-0.001, relative to wild-type GH activity (a)
additional information
NAD+
-
mutant V455A, kcat = 0.254 +/-0.001, relative to wild-type GH activity (a)
additional information
NAD+
relative kcat = 1.00 +/-0.02, wild-type GH activity
additional information
NAD+
-
relative kcat = 1.00 +/-0.02, wild-type GH activity
additional information
NAD+
relative kcat = 1.00 +/-0.05, wild-type GH activity (a) and (b)
additional information
NAD+
-
relative kcat = 1.00 +/-0.05, wild-type GH activity (a) and (b)
additional information
NAD+
relative kcat = 1.00 +/-0.09, wild-type ADPRT activity (A)-(D), and (a) and (b)
additional information
NAD+
-
relative kcat = 1.00 +/-0.09, wild-type ADPRT activity (A)-(D), and (a) and (b)
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
E553A
impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members
G454A
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
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+
R551A
ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)
G454A
reduced ADPRT activity compared to wild-type, Gly454 is part of active-site loop 1 (453-463)
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
drug development
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Lee, H.; Iglewski, W.J.
Cellular ADP-ribosyltransferase with the same mechanism of action as diphtheria toxin and Pseudomonas toxin A
Proc. Natl. Acad. Sci. USA
81
2703-2707
1984
Bos taurus, Mesocricetus auratus, Pseudomonas aeruginosa
Sanai, Y.; Morihara, K.; Tsuzuki, H.; Homma, J.Y.; Kato, I.
Proteolytic cleavage of exotoxin A from Pseudomonas aeruginosa: formation of an ADP-ribosyltransferase active fragment by the action of Pseudomonas elastase
FEBS Lett.
120
131-134
1980
Pseudomonas aeruginosa
Leppla, S.H.; Martin, O.C.; Muehl, L.A.
The exotoxin P. aeruginosa: a proenzyme having an unusual mode of activation
Biochem. Biophys. Res. Commun.
81
532-538
1978
Pseudomonas aeruginosa
Armstrong, S.; Merrill, A.R.
Toward the elucidation of the catalytic mechanism of the mono-ADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A
Biochemistry
43
183-194
2004
Pseudomonas aeruginosa
Zhang, Y.; Liu, S.; Lajoie, G.; Merrill, A.R.
The role of the diphthamide-containing loop within eukaryotic elongation factor 2 in ADP-ribosylation by Pseudomonas aeruginosa exotoxin A
Biochem. J.
413
163-174
2008
Pseudomonas aeruginosa
Jorgensen, R.; Wang, Y.; Visschedyk, D.; Merrill, A.R.
The nature and character of the transition state for the ADP-ribosyltransferase reaction
EMBO Rep.
9
802-809
2008
Pseudomonas aeruginosa (P11439), Pseudomonas aeruginosa
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