TAILIEUCHUNG - Báo cáo khoa học: Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp. BH72

Nitrogenase Fe-protein modification was analyzed in the endophyticb-pro-teobacterium Azoarcussp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. | Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp. BH72 Janina Oetjen1 Sascha Rexroth2 and Barbara Reinhold-Hurek1 1 GeneralMicrobiology Faculty of Biology and Chemistry University Bremen Germany 2 Plant Biochemistry Faculty of Biology and Biotechnology Ruhr University Bochum Germany Keywords ADP-ribosylation Azoarcus sp. BH72 mass spectrometry nitrogenase post-translational modification Correspondence B. Reinhold-Hurek GeneralMicrobiology Faculty of Biology and Chemistry University Bremen Postfach 33 04 40 D-28334 Bremen Germany Fax 49 0 421 218 9058 Tel 49 0 421 218 2370 E-mail breinhold@ Received 20 February 2009 revised 16 April 2009 accepted 1 May 2009 doi Nitrogenase Fe-protein modification was analyzed in the endophytic b-pro-teobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosy-lations. Catalyzing the reduction approximately 300 X .

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