Mass-spectrometric characterization of two posttranslational modifications of cysteine dioxygenase |
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Authors: | Torsten Kleffmann Seino A K Jongkees Graham Fairweather Sigurd M Wilbanks Guy N L Jameson |
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Institution: | (1) Department of Biochemistry, Centre for Protein Research, University of Otago, PO Box 56, Dunedin, 9054, New Zealand;(2) Department of Biochemistry, University of Otago, PO Box 56, Dunedin, 9054, New Zealand;(3) Department of Chemistry, University of Otago, PO Box 56, Dunedin, 9054, New Zealand |
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Abstract: | Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent
to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was
variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields “mature” and “immature”
forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the
products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The
cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage
by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry.
Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating
agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier.
Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small
amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation,
they may actually arise via nonenzymatic, nonphysiological processes.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | Cysteine dioxygenase Oxygen Iron Cystine Non-heme monoiron |
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