Lysine and polyamines are substrates for transglutamination of Rho by the Bordetella dermonecrotic toxin

Bordetella dermonecrotic toxin (DNT) catalyzes the transglutamination of gl utamine-63/61 of Rho GTPases, thereby constitutively activating Rho protein s. Here we identified second substrates for transgutamination of RhoA by DI NT. The enzymatically active fragment of DNT (residues 1136 to 1451, Delta DNT) induced the incorporation of L-[C-14]lysine in RhoA in a concentration -dependent manner. Also, Rac and Cdc42, but not Ras, were transglutaminated with lysine by Delta DNT. Transglutamination of the GTPase With L-lysine i nhibited intrinsic and Rho-GAP-stimulated GTP hydrolysis of RhoA. In contra st to lysine, treatment of RhoA with alanine, arginine, and glutamine were not able to substitute for lysine in the transglutamination reaction. DNT i ncreased the incorporation of L-[C-14]lysine into embryonic bovine lung cel ls. Microinjection of GST-RhoA together with the enzymatically active DNT f ragment into Xenopus oocytes, subsequent affinity purification of modified GST-RhoA, and mass spectrometry identified attachment of putrescine or sper midine at glutamine-63 of RhoA. A comparison of putrescine, spermidine, and lysine as substrates for DNT-induced transglutamination of RhoA revealed t hat lysine is a preferred second substrate at least in vitro.