Purification and functional characterization of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii

D. Jahn, Y. C. Kim, Y. Ishino, M. W. Chen, D. Soll

研究成果: Contribution to journalArticle査読

37 被引用数 (Scopus)

抄録

The formation of glutaminyl-tRNA (Gln-tRNA) in Bacilli, chloroplasts, and mitochondria occurs in a two-step reaction. This involves misacylation of tRNA(Gln) with glutamate by glutamyl-tRNA synthetase and subsequent amidation of Glu-tRNA(Gln) to the correctly acylated Gln-tRNA(Gln) by a specific amidotransferase (Schon, A., Kannangara, C.G., Gough, S., and Soll, D. (1988) Nature 331, 187-190). Here we demonstrate the existence of this pathway in green algae and describe the purification of the Glu-tRNA(Gln) amidotransferase from Chlamydomonas reinhardtii. The purified enzyme showed an M(r) of approximately 120,000 when analyzed by glycerol gradient sedimentation and gel filtration. An apparent M(r) of 63,000 of the denatured protein was demonstrated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. This indicates that the enzyme possesses an α2 structure. The substrate for the purified enzyme is Glu-tRNA(Gln) but not Glu-tRNA(Glu). The enzyme requires ATP, Mg2+, and an amide donor for the conversion. Acceptable amide donors are glutamine, asparagine, and ammonia. Blocking of the glutamine-dependent reaction by alkylation of the protein with 6-diazo-5-oxonorleucine did not inhibit the ammonia-dependent reaction, suggesting that the enzyme has separate glutamine and ammonia binding sites. As suggested by Wilcox (Wilcox, M. (1969) Eur. J. Biochem. 11, 405-412) the amidation reaction may involve glutamyl-phosphate formation, since ATP is cleaved to ADP when the enzyme is incubated with Glu-tRNA(Gln) and ATP. In common with other glutamine amidotransferases, the enzyme also possesses low glutaminase activity. The purified Glu-tRNA(Gln) amidotransferase forms a stable complex with Glu-tRNA(Gln) in the presence of ATP and Mg2+ but in the absence of the amide donor as determined by gradient centrifugation.

本文言語英語
ページ(範囲)8059-8064
ページ数6
ジャーナルJournal of Biological Chemistry
265
14
出版ステータス出版済み - 1990
外部発表はい

All Science Journal Classification (ASJC) codes

  • 生化学
  • 分子生物学
  • 細胞生物学

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