2.4.2.36: NAD+-diphthamide ADP-ribosyltransferase

part of active-site loop 1 (453-463)

D463A

part of active-site loop 1 (453-463)

E546A

reduced ADPRT activity compared to wild-type possibly due to leakage of aqueous solvent into binding pocket which prevents ADP-ribose transfer, no impaired NAD+ binding and glycohydrolase activity, Glu546 crucial for ADPRT activity of ExoA but not strictly conserved among toxin family members, part of active-site loop 3 (546-551), upon NAD+-binding Glu546 and Tyr481 (both connected by hydrogen bonds resulting in water molecule exclusion) in hydrogen bonding distance to nucleophilic N3 of diphthamide imidazole possibly increasing its nucleophilic character

E546A/R551A

double alanine mutant, almost complete loss of ADPRT activity not restored by mutations E546D/R551K or E546R/R551E

E546D

lesser ADPRT activity than E546A

E546F

part of active-site loop 3 (546-551)

E546H

see E546A

E546N

partial rescue of ADPRT activity compared to E546A

E546Q

part of active-site loop 3 (546-551)

E547A

part of active-site loop 3 (546-551)

E548A

part of active-site loop 3 (546-551)

E553A

impaired NAD+ binding and glycohydrolase activity, Glu553 is a crucial catalytic residue and conserved among toxin family members

G453A

part of active-site loop 1 (453-463)

G454A

2 entries

G549A

part of active-site loop 3 (546-551)

G550A

part of active-site loop 3 (546-551)

L462A

part of active-site loop 1 (453-463)

L552A

QuickChange mutagenesis

Q460A

reduced ADPRT activity compared to wild-type, Gln460 is part of active-site loop 1 (453-463), active-site loop1 flips towards diphthamide of eEF2 by a hinged action of Ala457 and Ala464 upon NAD+ binding which places Gln460 close to adenine phosphate of NAD+, interaction of Gln460 with A-phosphate of NAD+ possibly prevents hydrolysis of NAD+ to AMP or ADP

R456A

part of active-site loop 1 (453-463)

R458A

56-fold reduction in ADPRT activity, 53-fold reduction in GH activity, and 31-fold increased KD for NAD+ compared to wild-type, ADPRT activity sensitivity towards substitution in order Gln>Lys>Trp>Ala>His, Arg458 is part of active-site loop 1 (453-463) and implicated in NAD+ substrate docking and orientation, active-site loop1 flips towards diphthamide of eEF2 by a hinged action of Ala457 and Ala464 upon NAD+ binding which enables van der Waals interactions between Arg458 and the adenine base of NAD+

R458H

part of active-site loop 1 (453-463)

R458K

part of active-site loop 1 (453-463)

R458Q

part of active-site loop 1 (453-463)

R458W

part of active-site loop 1 (453-463)

R551A

ADPRT activity sensitive to replacements in order Ala>Lys>Gln>Glu>His>Cys, Arg551 is part of active-site loop 3 (546-551)

R551C

part of active-site loop 3 (546-551)

R551E

part of active-site loop 3 (546-551)

R551H

part of active-site loop 3 (546-551)

R551K

part of active-site loop 3 (546-551)

R551Q

part of active-site loop 3 (546-551)

S459A

part of active-site loop 1 (453-463)

V455A

part of active-site loop 1 (453-463)

DELTA424-634

catalytic domain of cholix has much greater affinity for NAD+ than the full-length enzyme. 15fold higher ADP-ribosyltransferase activity than full-length enzyme

E574A

mutation of catalytic fragment DELTA424-634: mutant does not have a large impact on NAD+-binding. Catalytic activity is 3fold reduced compared to catalytic fragment DELTA424-634

E574A/E581A

mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity

E579R

mutation of catalytic fragment DELTA424-634: NAD+ binding not changed significantly. Catalytic activity is 1.6fold reduced compared to catalytic fragment DELTA424-634

E581A

mutation of catalytic fragment DELTA424-634: mutant has low affinity for NAD+. Mutant shows no catalytic activity

Y493A

mutation of catalytic fragment DELTA424-634: mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant shows a marked reduction in activity compared to catalytic fragment DELTA424-634

Y504A

mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant shows a marked reduction in activity compared to catalytic fragment DELTA424-634

Y504F

mutation of catalytic fragment DELTA424-634: mutant has little effect on NAD+-binding. Mutant has lower affinity for NAD+ than Y504A mutant. Mutant shows a mild reduction in activity compared to catalytic fragment DELTA424-634

additional information