ADP-Ribosylargininyl reaction of cholix toxin is mediated through diffusible intermediates Reportar como inadecuado




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BMC Biochemistry

, 15:26

Chemical biology

Abstract

BackgroundCholix toxin is an ADP-ribosyltransferase found in non-O1-non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.

ResultsOur studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.

ConclusionsWe discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD intermediate was proposed to react with arginine residues in a proximity dependent manner.

KeywordsCholix toxin ADP-ribosylation Auto-ADP-ribosylation Arginine AbbreviationsCTcCholix toxin catalytic fragment

CXTFull-length cholix toxin

hADPRHHuman ADP-ribosylarginine hydrolase

Ig-CTcCTc fused to an immunoglobulin constant domain

ethenoNADNicotinamide 1,N-ethenoadenine dinucleotide

ethenoAMP1,N-ethenoadenosine 5′-monophosphate disodium salt

biotinyl-NAD6-biotin-7-NAD

ADP-riboseAdenosine 5′-diphosphoribose

Electronic supplementary materialThe online version of this article doi:10.1186-s12858-014-0026-1 contains supplementary material, which is available to authorized users.

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Autor: Vicky M-H Sung - Chia-Lun Tsai

Fuente: https://link.springer.com/







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