BRENDA - Enzyme Database
show all sequences of 2.4.2.36

The nature and character of the transition state for the ADP-ribosyltransferase reaction

Jorgensen, R.; Wang, Y.; Visschedyk, D.; Merrill, A.R.; EMBO Rep. 9, 802-809 (2008)

Data extracted from this reference:

Application
Application
Commentary
Organism
drug development
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
Pseudomonas aeruginosa
Crystallization (Commentary)
Crystallization
Organism
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
Pseudomonas aeruginosa
Engineering
Amino acid exchange
Commentary
Organism
D461A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
D463A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
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
Pseudomonas aeruginosa
E546A/R551A
double alanine mutant, almost complete loss of ADPRT activity not restored by mutations E546D/R551K or E546R/R551E
Pseudomonas aeruginosa
E546D
lesser ADPRT activity than E546A
Pseudomonas aeruginosa
E546F
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E546H
see E546A
Pseudomonas aeruginosa
E546N
partial rescue of ADPRT activity compared to E546A
Pseudomonas aeruginosa
E546Q
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E547A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E548A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E553A
impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members
Pseudomonas aeruginosa
G453A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
G454A
part of active-site loop 1 (453-463); reduced ADPRT activity compared to wild-type, Gly454 is part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
G549A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
G550A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
L462A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
L552A
QuickChange mutagenesis
Pseudomonas aeruginosa
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
Pseudomonas aeruginosa
R456A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
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+
Pseudomonas aeruginosa
R458H
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458K
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458Q
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458W
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R551A
ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551C
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551E
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551H
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551K
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551Q
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
S459A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
V455A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
NAD+
double mutant E546A/R551A, relative KD = 1.11 +/-0.03; double mutant E546D/R551K, relative KD = 2.71 +/-0.74; double mutant E546R/R551E, relative KD = 1.29 +/-0.49; mutant D461A, KD = 1.03 +/-0.02 relative to wild-type (c); mutant D463A, KD = 1.18 +/-0.04 relative to wild-type (c); mutant E546A, relative KD = 1.77 +/-0.34; mutant E546D, relative KD = 3.83 +/-0.89; mutant E546F, relative KD = 1.97 +/-0.11; mutant E546H, relative KD = 1.03 +/-0.09; mutant E546N, relative KD = 2.14 +/-0.26; mutant E546Q, relative KD = 2.31 +/-0.71; mutant E547A, relative KD = 1.97 +/-0.77; mutant E548A, relative KD = 2.69 +/-0.11; mutant E553A, relative KD = 2.57 +/-0.86; mutant G453A, KD = 4.85 +/-1.07 relative to wild-type (c); mutant G454A, KD = 6.25 +/-0.22 relative to wild-type (c); mutant G549A, relative KD = 8.00 +/-0.94; mutant G550A, relative KD = 6.60 +/-0.54; mutant L462A, KD = 1.86 +/-0.10 relative to wild-type (c); mutant L552A, relative KD = 4.43 +/-0.57; mutant Q460A, KD = 4.30 +/-0.43 relative to wild-type (c); mutant R456A, KD = 3.56 +/-0.53 relative to wild-type (c); mutant R458A, KD = 5.78 +/-0.18 relative to wild-type (c); mutant R458H, KD = 31.02 +/-7.18 relative to wild-type (d); mutant R458K, KD = 3.18 +/-0.23 relative to wild-type (c); mutant R458Q, KD = 0.45 +/-0.06 relative to wild-type (e); mutant R458W, KD = 11.26 +/-1.16 relative to wild-type (d); mutant R551A, relative KD = 2.69 +/-0.03; mutant R551C, relative KD = 5.57 +/-0.91; mutant R551E, relative KD = 5.03 +/-0.51; mutant R551H, relative KD = 2.29 +/-0.46; mutant R551K, relative KD = 1.29 +/-0.03; mutant R551Q, relative KD = 2.63 +/-0.39; mutant S459A, KD = 1.59 +/-0.16 relative to wild-type (c); mutant V455A, KD = 3.31 +/-0.24 relative to wild-type (c); wild-type, KD = 35 +/-3 microM, relative KD = 1.00 +/-0.09, estimated according to quenched intrinsic tryptophan fluorescence upon NAD+ binding to the active site; wild-type, KD = 36 +/-8 microM (c), KD = 62 +/-2 microM (d), KD = 74 +/-16 microM, relative KD = 1.00 +/-0.03
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
additional information
Pseudomonas aeruginosa
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
?
-
-
-
NAD+ + essential ribosomal elongation factor 2
Pseudomonas aeruginosa
transfer of ADP-ribose moiety (oxocarbenium ion) of NAD+ onto N3 of diphthamide imidazole of essential ribosomal elongation factor 2 (eEF2)
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Pseudomonas aeruginosa
P11439
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
692077
Pseudomonas aeruginosa
?
-
-
-
-
additional information
no ADP-ribosyl transfer by mutants R551H and R551C despite of occurring NAD+-binding and hydrolysis
692077
Pseudomonas aeruginosa
?
-
-
-
-
NAD+ + 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)
692077
Pseudomonas aeruginosa
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
-
?
NAD+ + essential ribosomal elongation factor 2
essential ribosomal elongation factor 2: eEF2, 2-step reaction by concerted GH and ADPRT activity, transition-state model
692077
Pseudomonas aeruginosa
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
-
?
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
additional information
-
NAD+
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)
Pseudomonas aeruginosa
Application (protein specific)
Application
Commentary
Organism
drug development
more potent therapeutics for treatment of bacterial diseases and infections through understanding of ADPRT reaction meachanism
Pseudomonas aeruginosa
Crystallization (Commentary) (protein specific)
Crystallization
Organism
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
Pseudomonas aeruginosa
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
D461A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
D463A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
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
Pseudomonas aeruginosa
E546A/R551A
double alanine mutant, almost complete loss of ADPRT activity not restored by mutations E546D/R551K or E546R/R551E
Pseudomonas aeruginosa
E546D
lesser ADPRT activity than E546A
Pseudomonas aeruginosa
E546F
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E546H
see E546A
Pseudomonas aeruginosa
E546N
partial rescue of ADPRT activity compared to E546A
Pseudomonas aeruginosa
E546Q
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E547A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E548A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
E553A
impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members
Pseudomonas aeruginosa
G453A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
G454A
part of active-site loop 1 (453-463); reduced ADPRT activity compared to wild-type, Gly454 is part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
G549A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
G550A
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
L462A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
L552A
QuickChange mutagenesis
Pseudomonas aeruginosa
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
Pseudomonas aeruginosa
R456A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
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+
Pseudomonas aeruginosa
R458H
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458K
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458Q
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R458W
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
R551A
ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551C
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551E
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551H
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551K
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
R551Q
part of active-site loop 3 (546-551)
Pseudomonas aeruginosa
S459A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
V455A
part of active-site loop 1 (453-463)
Pseudomonas aeruginosa
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
NAD+
double mutant E546A/R551A, relative KD = 1.11 +/-0.03; double mutant E546D/R551K, relative KD = 2.71 +/-0.74; double mutant E546R/R551E, relative KD = 1.29 +/-0.49; mutant D461A, KD = 1.03 +/-0.02 relative to wild-type (c); mutant D463A, KD = 1.18 +/-0.04 relative to wild-type (c); mutant E546A, relative KD = 1.77 +/-0.34; mutant E546D, relative KD = 3.83 +/-0.89; mutant E546F, relative KD = 1.97 +/-0.11; mutant E546H, relative KD = 1.03 +/-0.09; mutant E546N, relative KD = 2.14 +/-0.26; mutant E546Q, relative KD = 2.31 +/-0.71; mutant E547A, relative KD = 1.97 +/-0.77; mutant E548A, relative KD = 2.69 +/-0.11; mutant E553A, relative KD = 2.57 +/-0.86; mutant G453A, KD = 4.85 +/-1.07 relative to wild-type (c); mutant G454A, KD = 6.25 +/-0.22 relative to wild-type (c); mutant G549A, relative KD = 8.00 +/-0.94; mutant G550A, relative KD = 6.60 +/-0.54; mutant L462A, KD = 1.86 +/-0.10 relative to wild-type (c); mutant L552A, relative KD = 4.43 +/-0.57; mutant Q460A, KD = 4.30 +/-0.43 relative to wild-type (c); mutant R456A, KD = 3.56 +/-0.53 relative to wild-type (c); mutant R458A, KD = 5.78 +/-0.18 relative to wild-type (c); mutant R458H, KD = 31.02 +/-7.18 relative to wild-type (d); mutant R458K, KD = 3.18 +/-0.23 relative to wild-type (c); mutant R458Q, KD = 0.45 +/-0.06 relative to wild-type (e); mutant R458W, KD = 11.26 +/-1.16 relative to wild-type (d); mutant R551A, relative KD = 2.69 +/-0.03; mutant R551C, relative KD = 5.57 +/-0.91; mutant R551E, relative KD = 5.03 +/-0.51; mutant R551H, relative KD = 2.29 +/-0.46; mutant R551K, relative KD = 1.29 +/-0.03; mutant R551Q, relative KD = 2.63 +/-0.39; mutant S459A, KD = 1.59 +/-0.16 relative to wild-type (c); mutant V455A, KD = 3.31 +/-0.24 relative to wild-type (c); wild-type, KD = 35 +/-3 microM, relative KD = 1.00 +/-0.09, estimated according to quenched intrinsic tryptophan fluorescence upon NAD+ binding to the active site; wild-type, KD = 36 +/-8 microM (c), KD = 62 +/-2 microM (d), KD = 74 +/-16 microM, relative KD = 1.00 +/-0.03
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
additional information
Pseudomonas aeruginosa
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
?
-
-
-
NAD+ + essential ribosomal elongation factor 2
Pseudomonas aeruginosa
transfer of ADP-ribose moiety (oxocarbenium ion) of NAD+ onto N3 of diphthamide imidazole of essential ribosomal elongation factor 2 (eEF2)
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
?
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
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
692077
Pseudomonas aeruginosa
?
-
-
-
-
additional information
no ADP-ribosyl transfer by mutants R551H and R551C despite of occurring NAD+-binding and hydrolysis
692077
Pseudomonas aeruginosa
?
-
-
-
-
NAD+ + 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)
692077
Pseudomonas aeruginosa
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
-
?
NAD+ + essential ribosomal elongation factor 2
essential ribosomal elongation factor 2: eEF2, 2-step reaction by concerted GH and ADPRT activity, transition-state model
692077
Pseudomonas aeruginosa
nicotinamide + H+ + ADP-ribosylated essential ribosomal elongation factor 2
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
additional information
-
NAD+
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)
Pseudomonas aeruginosa
Other publictions for EC 2.4.2.36
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [C]
Temperature Range [C]
Temperature Stability [C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [C] (protein specific)
Temperature Range [C] (protein specific)
Temperature Stability [C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
737282
Lugo
The father, son and cholix tox ...
Pseudomonas aeruginosa, Vibrio cholerae
Toxins
7
2757-2772
2015
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722762
Fieldhouse
The 1.8 A cholix toxin crystal ...
Vibrio cholerae
J. Biol. Chem.
287
21176-21188
2012
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699495
Jang
Improved purification process ...
Vibrio cholerae, Vibrio cholerae 569B
J. Microbiol. Biotechnol.
19
108-112
2009
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701050
Gong
Cloning, expression, purificat ...
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Protein Expr. Purif.
191
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2009
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690825
Zhang
The role of the diphthamide-co ...
Pseudomonas aeruginosa
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413
163-174
2008
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692077
Jorgensen
The nature and character of th ...
Pseudomonas aeruginosa
EMBO Rep.
9
802-809
2008
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696625
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Design, synthesis, and evaluat ...
Vibrio cholerae
Bioorg. Med. Chem. Lett.
18
3724-3727
2008
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698219
Feng
Investigation of the role of c ...
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Immunol. Invest.
37
782-797
2008
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658054
Armstrong
Toward the elucidation of the ...
Pseudomonas aeruginosa
Biochemistry
43
183-194
2004
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489798
Lee
Cellular ADP-ribosyltransferas ...
Bos taurus, Mesocricetus auratus, Pseudomonas aeruginosa
Proc. Natl. Acad. Sci. USA
81
2703-2707
1984
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489799
Sanai
Proteolytic cleavage of exotox ...
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FEBS Lett.
120
131-134
1980
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489800
Leppla
The exotoxin P. aeruginosa: a ...
Pseudomonas aeruginosa
Biochem. Biophys. Res. Commun.
81
532-538
1978
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